- Email: [email protected]
MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin
E XP ER I ME NTAL C E LL RE S E ARCH
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
] (]]]]) ]]]–]]]
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin Q1
Yichen Songa, Ping Wangb,c, Wei Zhaob,c, Yilong Yaoa, Xiaobai Liud, Jun Mab,c, Yixue Xueb,c, Yunhui Liua,n a
Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang 110004, People's Republic of China Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang 110001, People's Republic of China c Institute of Pathology and Pathophysiology, China Medical University, Shenyang 110001, People's Republic of China d The 96th Class, 7-year Program, China Medical University, Shenyang, Liaoning Province 110001, People's Republic of China b
article information
abstract
Article Chronology:
MiR-17-92 cluster has recently been reported as an oncogene in some tumors. However, the
Received 5 November 2013
association of miR-18a, an important member of this cluster, with glioblastoma remains
Received in revised form
unknown. Therefore, this study aims to investigate the expression of miR-18a in glioblastoma
5 March 2014
and its role in biological behavior of U87 and U251 human glioblastoma cell lines. Quantitative
Accepted 10 March 2014
RT-PCR results showed that miR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines compared with that in human brain tissues and primary normal human
Keywords:
astrocytes, and the expression levels were increased along with the rising pathological grades
Glioblastoma
of glioblastoma. Neogenin was identified as the target gene of miR-18a by dual-luciferase reporter
MiR-18a
assays. RT-PCR and western blot results showed that its expression levels were decreased along
Neogenin
with the rising pathological grades of glioblastoma. Inhibition of miR-18a expression was
Proliferation
established by transfecting exogenous miR-18a inhibitor into U87 and U251 cells, and its effects
Migration
on the biological behavior of glioblastoma cells were studied using CCK-8 assay, transwell assay
Invasion
and flow cytometry. Inhibition of miR-18a expression in U87 and U251 cells significantly upregulated neogenin, and dramatically suppressed the abilities of cell proliferation, migration and invasion, induced cell cycle arrest and promoted cellular apoptosis. Collectively, these results suggest that miR-18a may regulate biological behavior of human glioblastoma cells by targeting neogenin, and miR-18a can serve as a potential target in the treatment of glioblastoma. & 2014 Published by Elsevier Inc.
Introduction Glioblastoma is one of the most common primary malignant tumors in the adult central nervous system. Although treatment
strategies of surgery, radiation and chemotherapy have been well developed in recent years, patients with glioblastoma show no significantly improved prognosis and the median survival is reported to be only 14 months [1,2]. Therefore, glioblastoma
n
Corresponding author. E-mail addresses: [email protected] (Y. Song), [email protected] (P. Wang), [email protected] (W. Zhao), [email protected] (Y. Yao), [email protected] (X. Liu), [email protected] (J. Ma), [email protected] (Y. Xue), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.yexcr.2014.03.009 0014-4827/& 2014 Published by Elsevier Inc.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
94 95 96 97 98 99 100 101 102 103 104 105
2
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
E XP E RI ME N TAL CE L L R ES E ARC H
remains one of the most intractable diseases in the field of neurosurgery. MicroRNAs (miRNAs) are a class of about 20–22 bp small noncoding RNAs. They act either as oncogenes or tumor suppressors, and regulate gene expression post-transcriptionally by binding with 30 -UTR which causes either degradation or repression of target mRNAs [3,4]. Many miRNAs were shown to be abnormally expressed in glioblastoma, and were involved in the development of glioblastoma [4,5]. It was reported that miR-21 was strongly overexpressed in human glioblastoma and could induce the deterioration of glioblastoma by blocking the expression of apoptosis-related genes [6,7]. Recently, it has been reported that miR-221/222 overexpression promoted the aggressive growth of human glioblastoma by regulating the cell cycle related factor p27 [8]. In addition, miR-128 and miR-34a also acted as tumor suppressors in glioblastoma [9,10]. Therefore, different miRNAs may produce different biological effects on glioblastoma. MiR-17-92 cluster, recognized as an oncogene family, was overexpressed in anaplastic thyroid cancer and colorectal tumors and might be involved in controlling the corresponding target genes to regulate the development of these tumors [11,12]. It has been reported that miR-17-92 might play a significant role in the development of glioblastoma since its expression was up-regulated in the tumor [13]. MiR-18a, an important member of miR-17-92 cluster, plays a carcinogenic role in some tumors. For example, it promoted the proliferation of hepatoma cells by inhibiting ERalpha expression, and acted as oncogene during gastric adenocarcinogenesis by negatively regulating the expression of protein inhibitor against activated signal transducer and activator of transcription 3 (STAT3) [14,15]. At present, the effects of miR-18a on glioblastoma were still rarely reported. Turchi et al. [16] demonstrated the contribution of miR-18an to the promotion of glioma initiating cells self-renewal and tumorigenicity. On the contrary, Fox et al. [17] found that miR-18a was correlated with improved survival in glioblastoma patients by regulating transforming growth factor-β signal transduction and connective tissue growth factor expression. Therefore, the function and molecular mechanism of miR-18a in human glioblastoma are still unclear. Neogenin, a homolog of Deleted in Colorectal Cancer (DCC), displays wide distribution and lifelong stable expression in the central nervous system as a dependent receptor of neurogenesisrelated proteins like netrin, or members of the repulsive guidance molecule family [18]. Similar to DCC, neogenin binds with its ligands to play various physiological roles in normal tissues, including axon guidance, cell migration, tissue morphogenesis and differentiation, angiogenesis, signal transduction and cellular apoptosis [18–20]. DCC and its homolog neogenin have been considered as tumor suppressors in a variety of tumors since loss of DCC expression was discovered in colorectal cancers [21–24]. Recent studies showed that neogenin expression was downregulated in lung adenocarcinomas and breast cancer, and the low expressed neogenin could accelerate the tumor progression [25,26]. In the central nervous system, neogenin acted as a tumor suppressor and was lowly expressed in glioblastoma compared with control brain tissues [27]. As previously mentioned, miR-18a could affect the tumor development by regulating the expression of related genes; however, whether miR-18a has regulatory effects on neogenin in glioblastoma remains unclear. Under such background, the major aim of this study was to investigate the regulatory effects of miR-18a on neogenin and biological behavior of glioblastoma cells.
] (]]]]) ]]]–]]]
Material and methods Clinical specimens Glioblastoma specimens and their homologous surrounding nonneoplastic tissues were obtained from the Department of Neurosurgery, Shengjing Hospital of China Medical University, China, from September 2012 to July 2013. Fresh human brain lobectomy specimens obtained from surgeries of malignant intracranial hypertension such as severe intracerebral hemorrhage or craniocerebral injury were used as negative control. All specimens were immediately frozen and preserved in liquid nitrogen following surgical resection. Glioma specimens were classified into four grades by two experienced clinical pathologists according to the 2007 WHO classification of tumors in the central nervous system, then they were divided into two groups: low-grade glioma group (WHO I–II, n¼ 3) and high-grade glioma group (WHO III–IV, n¼3). The research methods in our study were approved by the Institutional Review Board at the Shengjing Hospital of China Medical University, China. All participants provided their written informed consent, and the hospital ethical committee approved the experiments.
Reagents and cell culture High-glucose Dulbecco's modified Eagle medium (DMEM), Dulbecco's modified Eagle medium/F12 mixed medium and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). Trizol, Opti-MEMs I and Lipofectamine2000 transfection reagents were purchased from Invitrogen (Carlsbad, CA, USA). MicroRNA-18a inhibitor and its negative control, pmirGLOneogenin 30 -UTR and pmirGLO-neogenin 30 -UTR-MUT dualluciferase miRNA target expression vectors, human neogenin with or without its 30 -UTR expression vectors pGCMV-neogenin-30 -UTR and pGCMV-neogenin were synthesized from GenePharma (Shanghai, China). pRI-CMV/GFP-hsa-miR-18a and its negative control were synthesized from Inovogen (Beijing, China). All other reagents and chemicals were purchased from Sigma-Aldrich (Shanghai, China). Human glioblastoma cell lines U87 and U251, and human embryonic kidney cell line HEK-293 were obtained from the Chinese Academy of Medical Sciences (Beijing, China). U87 and HEK-293 cells were cultured in high glucose DMEM medium supplemented with 10% FBS, and U251 cells were cultured in DMEM/F12 medium supplemented with 10% FBS. All cells were maintained in a humidified incubator at 37 1C with 5% CO2. Primary normal human astrocytes (NHA) were purchased from the Sciencell Research Laboratories (Carlsbad, CA, USA) and cultured under the instructed condition by the manufacturer.
Cell transfection Glioma cells at approximately 50–70% confluence were transfected using Opti-MEMs I and Lipofectamine2000 reagents (Invitrogen, CA, USA) after 24 h of culture. Transfection complexes and methods were prepared according to the manufacturer's instructions. MiR-18a inhibitor sequence: 50 -CUA UCU GCA CUA GAU GCA CCU UA-30 and miR-18a inhibitor negative control (NC) sequence: 50 -CAG UAC UUU UGU GUA GUA CAA-30 . After 6 h of transfection, the transfection medium was replaced with
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225
E X PE R IM EN TA L C ELL R E S EA RC H
226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
high-glucose DMEM or DMEM/F12 medium with 10% FBS, and the cells were incubated for additional 24–96 h, then the transfected cells were harvested for further studies. U87 and U251 cells transfected with miR-18a inhibitor or its negative control were divided into three groups according to the experimental requirement: untransfected group, miR-18a inhibitor NC group and miR18a inhibitor group. Meanwhile, U87 and U251 cells cotransfected with pRI-CMV/GFP-hsa-miR-18a or its negative control and pGCMV-neogenin, and pGCMV-neogenin-30 -UTR or their negative control were divided into four groups: miR-18a-conþempty vector (EV) group; miR-18aþEV group; miR18aþneogenin group; miR-18aþneogenin 30 -UTR group.
Bioinformatics prediction and luciferase reporter assay The common targets of miR-18a predicted by computer-aided algorithms were obtained from multiple target prediction programs: Targetscan and Miranda (http://www.targetscan.org/ and http:// www.microrna.org/). HEK-293 cells were seeded in 96-well plates for 24 h, then cells at 50–70% confluence were co-transfected with wild-type or mutated pmirGLO-neogenin 30 -UTR reporter plasmid and pRI-CMV/GFP-hsa-miR-18a or pRI-CMV/GFP-hsa-miR-18a-con. HEK-293 cells were divided into five groups: pmirGLO group (blank); miR-18aþpmirGLO-neogenin 30 -UTR group; miR-18aþ pmirGLO-neogenin 30 -UTR-MUT group; miR-18a-conþpmirGLO-neo genin 30 -UTR group; miR-18a-conþpmirGLO-neogenin 30 -UTR-MUT group. Luciferase activity assays were performed at 48 h after transfection using the dual-luciferase reporter assay system (Pro mega, Madison, WI, USA). Renilla luciferase activity was used as an internal control.
RNA isolation, RT-PCR and quantitative RT-PCR (qRT-PCR) Total RNA of each group was extracted from the tissues and NHA, U87, and U251 cells using Trizol according to the manufacturer's instructions. After extraction, total RNA was divided into two parts. One part was used for qRT-PCR analysis of miR-18a, using TaqMans MicroRNA RT kit (Applied Biosystems, Carlsbad, CA, USA) for reverse transcription, then using TaqMans Universal Master Mix II (Applied Biosystems) and ABI 7500 Fast Real-Time PCR System for qRT-PCR analysis. The reaction conditions: 50 1C for 2 min, 95 1C for 10 min, and 40 cycles of 95 1C for 15 s, and 60 1C for 1 min. The expression level of miR-18a was normalized with reference to expression level of U6, and fold changes were calculated by relative quantification (2 ΔΔCt). The primers of miR18a (Hs01932578_s1) and U6 (001973) were synthesized from the Applied Biosystems. The other part of total RNA was used to detect mRNA level of neogenin, according to RNA PCR Kit (AMV) Ver.3.0 (TaKaRa, Dalian, Liaoning Province China), RNA was reversed into cDNA, then amplified by PCR using the following primers: neogenin forward: 50 -GAC TCC CGA TAC TAC ACC GT-30 , reverse: 50 -AAT TGG ACA GCT TCA GAC ATG-30 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward: 50 -GGT GAA GGT CGG AGT CAA CG-30 , reverse 50 -CCA TGT AGT TGA GGT CAA TGA AG-30 . The reaction conditions: 35 cycles of 94 1C denaturation for 30 s, 55 1C annealing for 30 s, and 72 1C extension for 1 min. The products were electrophoresed using 1% agarose gel and stained with GeneFinder. The relative neogenin mRNA level was normalized to that of GAPDH mRNA level using Quality One analysis software (Bio-Rad Laboratories, Hercules, CA,
] (]]]]) ]]]–]]]
3
USA). The primers for RT-PCR were synthesized from Sangon Biotech (Shanghai, China).
Western blot analysis Total protein from tissues and cells was extracted in RIPA buffer (Beyotime, Haimen, Jiangsu Province, China) supplemented with 1% phenylmethyl sulfonylfluoride. The samples were centrifuged at 12,000g, 4 1C for 20 min and the protein concentration was determined by the BCA method (Beyotime). Equal amounts of protein samples were separated using 10% sodium dodecyl sulfate polyacrylamide gel electropheresis and then electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore, Shanghai, China). The membranes were blocked in blocking buffer (5% non-fat milk dissolved in Tris–buffered saline–Tween) overnight at 4 1C. The blots were then respectively incubated with rabbit polyclonal anti-neogenin (sc-15337) antibody (1:400; Santa Cruz Biotechnology, Dallas, Texas, USA) and mouse monoclonal anti-GAPDH (sc-47724) antibody (1:5 000; Santa Cruz Biotechnology) overnight at 4 1C. Then the blots were incubated with corresponding secondary antibody at room temperature for 2 h after four washes for 30 min. Finally, protein bands were visualized by ECL (Santa Cruz Biotechnology) and detected by ECL Detection Systems (Thermo Scientific, Beijing, China). The relative integrated density values were calculated based on GAPDH protein as an internal control.
Cell proliferation assay U87 and U251 cells were seeded in 96-well plates at the density of 2000 cells/well, and cell inhibitory rate was assayed using the Cell Counting Kit-8 (Beyotime) at 24, 48, 72, and 96 h after transfection. 10 μL CCK-8 solution was added into each well and cells were incubated for another 1.5 h in a humidified incubator. Optical density value was measured at 450 nm. Five replicate wells were set up in each group and repeated three independent experiments were performed.
Cell migration and invasion assay Migration and invasion of U87 and U251 cells in vitro were assayed using a Transwell chamber (Costar, Corning, NY, USA) with a polycarbonic membrane (6.5 mm in diameter, 8 μm pore size). In the migration assay, cellular medium was replaced with serum-free medium after 24 h of transfection and cells were incubated for another 24 h. Then cells were trypsinized and suspended into single cells with serum-free medium at the density of 5 105 cells/mL. 100 μL of the cell suspension was added to the upper chamber, and 600 mL of high-glucose DMEM or DMEM/F12 medium supplemented with 10% FBS was added to the lower chamber. Cells were incubated for 24 h at 37 1C, then non-migrating cells on the top surface of membrane were removed with cotton swabs. Cells that migrated to the lower surface of the membrane were then fixed with methanol and stained with 20% Giemsa solution for 30 min at 37 1C and washed twice with phosphate buffer saline (PBS). Then stained cells were observed under an inverted microscope (400 ) to count the cell number within five randomly chosen fields and the average number was calculated. In the invasion assay, the transwell membrane was coated with 80 mL of Matrigel solution
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345
4
346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405
E XP E RI ME N TAL CE L L R ES E ARC H
] (]]]]) ]]]–]]]
(500 ng/mL; BD, Franklin Lakes, NJ, USA) and incubated at 37 1C for 4 h, the remaining steps were similar to the migration assay.
Cell cycle analysis Cell cycle assay was performed at 48 h after transfection. Cells in each group were trypsinized and resuspended in ice-cold PBS and gently pelleted by centrifugation at 2000 rpm, 4 1C for 5 min, then the cell density was adjusted to 1 106 cells/mL with ice-cold PBS. After being centrifuged at 2000 rpm, 4 1C for 5 min, cells were fixed and permeabilized in 70% ethanol overnight at 20 1C. Fixed cells were washed with PBS and incubated with 100 μL RNase A solution in water bath at 37 1C for 30 min according to the instruction of Cell Cycle Assay (KeyGEN, Nanjing, Jiangsu Province, China), then 400 μL of propidium iodide (PI) staining solution was added and the system was kept in dark at 4 1C for 30 min. Finally, cells were analyzed for DNA content on BD FACSCalibur (BD Biosciences, San Jose, CA, USA).
Apoptosis detection The effect of miR-18a expression on cellular apoptosis was assessed by Annexin V-FITC/ PI double staining kit (Beyotime). At 48 h after transfection, cells were collected and stained with Annexin V-FITC and PI according to the manufacturer's instruction, then they were analyzed on BD FACSCalibur.
Statistical analysis All the above experiments were independently repeated three times. All results were expressed as mean7SD, and all data were analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA). Group comparisons were analyzed with one-way analysis of variance, with Po0.05 as statistically significant difference.
Results Up-regulation of miR-18a expression in glioblastoma tissues and cell lines The expression levels of miR-18a mRNA in control brain, lowgrade glioma, high-grade glioma tissues and NHA, U87, and U251 cells were analyzed by qRT-PCR. As shown in Fig. 1, miR-18a mRNA expression was significantly up-regulated in glioblastoma tissues and two glioblastoma cell lines compared with the average expression levels in control brain tissues and NHA (Po0.05). Furthermore, the expression levels were elevated with the rising pathological grades of glioblastoma.
Down-regulation of neogenin expression in glioblastoma tissues and cell lines The neogenin mRNA and protein expression levels in control brain tissues, glioblastoma tissues and NHA, U87, and U251 cells were analyzed using RT-PCR and western blot assays, respecticvely. The RT-PCR results (Fig. 2A) showed that, neogenin mRNA expression was significantly down-regulated in glioblastoma tissues and two glioblastoma cell lines compared with that in homologous surrounding non-neoplastic tissues and NHA (Po0.05). Furthermore,
Fig. 1 – MiR-18a was overexpressed in human glioblastoma tissues and cell lines. Expression level of miR-18a was assessed in control brain tissues, low-grade glioma tissues, high-grade glioma tissues and NHA, U87, and U251 cells by qRT-PCR. U6 was used as an internal control. Data were presented as mean7SD from three independent experiments. *Po0.05, vs. the control group and #Po0.05, vs. NHA.
the expression level was also lower in high-grade glioma tissues than in low-grade glioma tissues. The western blot analysis results (Fig. 2B) were similar to RT-PCR, while neogenin protein expression was negative in U87 and U251 cells.
MiR-18a inhibitor significantly up-regulated neogenin expression in glioblastoma cells The expression of miR-18a and neogenin in U87 and U251 cells transfected with exogenous miR-18a inhibitor was analyzed at 24, 48, 72, and 96 h after transfection. The qRT-PCR results showed that the expression level of miR-18a was lowest at 48 h after transfection (Fig. S1A), likewise, the expression levels of neogenin mRNA and protein were highest at 48 h after transfection (Fig. S1B). Therefore, the regulatory effects of miR-18a on neogenin expression in glioblastoma cells were detected at 48 h after transfection in the following experiments. After U87 and U251 cells were transfected for 48 h, the miR-18a expression was significantly decreased compared with that of miR-18a inhibitor NC group, while there was no obvious difference between untransfected group and miR-18a inhibitor NC group (Fig. 3A). Meanwhile, as shown in Figs. 3B and C, the mRNA and protein expressions of neogenin in U87 and U251 cells were significantly increased in miR-18a inhibitor group compared with that of miR18a inhibitor NC group; however, there was no apparent difference between untransfected group and miR-18a inhibitor NC group. These results suggested that miR-18a could negatively regulate neogenin mRNA and protein expressions in U87 and U251 cells.
MiR-18a targeted the neogenin 30 -UTR The target genes of miR-18a were identified through bioinformatics analyses available on the miRNA database (http://www.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
E X PE R IM EN TA L C ELL R E S EA RC H
466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525
] (]]]]) ]]]–]]]
5
Fig. 2 – Neogenin expression was significantly down-regulated in human glioblastoma tissues and cell lines. (A) The neogenin mRNA level was decreased in glioma tissues (T) compared with the homologous surrounding non-neoplastic tissues (S) (Po0.05). The neogenin mRNA level in U87 and U251 cells (C) was also decreased compared with that in NHA (C) (Po0.05). The expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B) The results of western blot analysis were similar to RT-PCR, while the neogenin in U87 and U251 cells was loss of expression. GAPDH was used as an internal control. Data was presented as mean7SD from three independent experiments. *Po0.05, vs. the homologous surrounding nonneoplastic tissues (S) and #Po0.05, vs. NHA (C).
Fig. 3 – MiR-18a negatively regulated the expression of neogenin in glioblastoma cells. (A) The expression level of miR-18a was assessed in transfected U87 and U251 cells. The results of qRT-PCR showed that miR-18a expression was decreased in miR-18a inhibitor group compared with its negative control group (Po0.05). U6 was used as an internal control. (B) The expression level of neogenin in U87 and U251 cells transfected with exogenous miR-18a inhibitor was changed. The RT-PCR results showed that the neogenin mRNA level was significantly up-regulated in miR-18a inhibitor group compared with its negative control group (Po0.05). (C) The western blot analysis results were similar to RT-PCR. Accompanying graphs show densitometry analysis of neogenin expression. Data was presented as mean7SD from three independent experiments. *Po0.05, vs. the miR-18a inhibitor NC group. Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585
6
586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645
E XP E RI ME N TAL CE L L R ES E ARC H
targetscan.org/ and http://www.microrna.org/). We found that the mRNA of neogenin was theoretically the target gene of miR-18a and its sequence has been maintained as highly bio-conservative in different species, such as the Homo sapiens, the Pan troglodytes , the Macaca mulatta etc. (Fig. 4A). So we speculated that miR-18a could target the 30 -UTR of neogenin. The wild- or mutated-type neogenin 30 -UTR was cloned into pmirGLO dual-luciferase miRNA target expression vector (Fig. 4B), and these constructs were validated by sequencing. Then the miR-18a, miR-18a-con and neogenin wild or mutated type luciferase reporter plasmids were co-transfected in HEK293 cells. Dual-Luciferases reporter assay system showed that, overex pressed miR-18a significantly decreased luciferase activity of wild-type pmirGLO-neogenin 30 -UTR compared with the miR-18a-con group, and miR-18a did not affect mutated pmirGLO-neogenin 30 -UTR-MUT luciferase activity (Fig. 4C). Collectively, these results indicated that neogenin might be the target of miR-18a.
MiR-18a inhibitor decreased the proliferation of glioblastoma cells The ability of cell proliferation was analyzed at 24, 48, 72, and 96 h after transfection (Fig. 5). The results demonstrated that the U87 and U251 cells transfected with miR-18a inhibitor exhibited a significant decrease of cell proliferation compared with miR-18a inhibitor NC group (Po0.05), while there was no apparent difference between untransfected group and miR-18a inhibitor NC group (P40.05; Fig. 5A). Furthermore, the inhibition rate of cell proliferation in miR-18a inhibitor group reached the maximum point at 48 h after transfection (Fig. 5B). In order to further validate the potential mechanism of biological behavior changes of glioblastoma cells related to the negative regulatory effects of miR-18a on neogenin, a rescue experiment was performed by co-transfecting miR-18a expression vector and pGCMV-neogenin or pGCMV-neogenin-30 -UTR in U87 and U251 cells. As shown in Fig. S2, the mRNA and protein expression levels of neogenin were
] (]]]]) ]]]–]]]
significantly down-regulated in miR-18aþneogenin 30 -UTR group compared with the miR-18aþneogenin group (Po0.05). And the results of the CCK-8 assay showed that the down-regulation of neogenin expression significantly promoted the proliferation of U87 and U251 cells (Fig. S3A).
MiR-18a inhibitor inhibited the migration and invasion of glioblastoma cells The migration and invasion of the U87 and U251 cells were examined at 48 h after transfection (Fig. 6). The results showed that the migration and invasion ability of U87 and U251 cells transfected with miR-18a inhibitor was significantly decreased compared with miR-18a inhibitor NC group (Po0.05), while there was no noticeable difference between the untransfected group and miR-18a inhibitor NC group (P40.05). The results of the rescue experiment showed that migration and invasion capacities of miR-18aþneogenin 30 -UTR group was obviously higher than that in miR-18aþneogenin group (Po0.05; Fig. S3B).
MiR-18a inhibitor induced G0/G1 arrest in glioblastoma cells The cell cycle in U87 and U251 cells was analyzed by flow cytometry at 48 h after transfection (Fig. 7). The results showed that the percentage of cells increased in G0/G1 phase and decreased in S and G2/M phase significantly in U87 and U251 cells transfected with miR-18a inhibitor compared with the miR18a inhibitor NC group (Po0.05), while there was no apparent difference between untransfected group and miR-18a inhibitor NC group. In the rescue experiment, the miR-18aþneogenin 30 -UTR group showed a lower percentage of cells in G0/G1 phase and a higher percentage of cells in S and G2/M phase compared with that in miR-18aþneogenin group (Po0.05; Fig. S3C).
Fig. 4 – MiR-18a negatively regulated neogenin expression through binding with neogenin 30 -UTR. (A) Schematic diagram of putative miR-18a binding site in the 30 -UTR of neogenin in human. The nucleotide sequence illustrates the predicted base-pairing between miR-18a and neogenin 30 -UTR. (B) The corresponding mutant nucleotides of the neogenin 30 -UTR was labeled in bold below. (C) MiR-18a down-regulated luciferase activities controlled by wild-type neogenin 30 -UTR, but did not affect luciferase activities controlled by mutant-type neogenin 30 -UTR, moreover, miR-18a-con did not affect luciferase activities controlled by wild-type or mutant-type neogenin 30 -UTR. Relative luciferase activities were calculated as the ratio of firefly/renilla activities in the cells and normalized to the pmirGLO group. Data were presented as mean7SD from three independent experiments. *Po 0.05, vs. the miR-18aþpmirGLO-neogenin 30 -UTR group. Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705
E X PE R IM EN TA L C ELL R E S EA RC H
706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765
] (]]]]) ]]]–]]]
7
Fig. 5 – MiR-18a inhibitor reduced cell proliferation in glioblastoma cells. CCK-8 assay showed that the U87 and U251 cells transfected with miR-18a inhibitor proliferated at lower levels than its negative control group (Po0.05). There was no obvious difference between untransfected group and miR-18a inhibitor NC group in the experiment. The U87 and U251 cells transfected for 48 h showed the highest inhibitory rate than the other three time point groups. Data were presented as mean7SD of three independent experiments. *Po0.05, vs. the miR-18a inhibitor NC group and #Po0.05, vs. the 24 h group.
Fig. 6 – MiR-18a inhibitor reduced migration and invasion in glioblastoma cells. U87 and U251 cells were transfected with miR-18a inhibitor and then subject to Transwell migration and invasion assays. After 24 h, migration and invasion cells were correspondingly counted after staining with 20% Giemsa. Representative photographs of migration and invasion cells on the membrane and accompanying statistical graphs were presented. The migration and invasion in U87 and U251 cells transfected with miR-18a inhibitor were significantly decreased compared with its negative control group (Po0.05). However, there was no obvious difference between untransfected group and miR-18a inhibitor NC group (P40.05). Data were presented as mean7SD of three independent experiments.*Po0.05, vs. the miR-18a inhibitor NC group. Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825
8
826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885
E XP E RI ME N TAL CE L L R ES E ARC H
] (]]]]) ]]]–]]]
Fig. 7 – MiR-18a inhibitor induced G0/G1 arrest in glioblastoma cells. The representative photographs showed cell cycle distribution of U87 and U251 cells in untransfected group, miR-18a inhibitor NC group and miR-18a inhibitor group. The percentage of cells in the different cell cycle phases was plotted, and we found that the percentage of cells transfected with miR-18a inhibitor distributed in G0/G1 phase was higher than its negative control group (Po0.05). And there was no obvious difference between untransfected group and miR-18a inhibitor NC group. Data were presented as mean7SD of three independent experiments. *Po0.05, vs. the miR-18a inhibitor NC group.
MiR-18a inhibitor promoted the apoptosis of glioblastoma cells The U87 and U251 cells were stained with Annexin V-FITC/PI and the apoptosis was determined by flow cytometry at 48 h after transfection (Fig. 8). The results demonstrated that the apoptosis rate in both cell lines transfected with miR-18a inhibitor was significantly increased compared with miR-18a inhibitor NC group (Po0.05), while there was no significant difference between untransfected group and miR-18a inhibitor NC group. In the rescue experiment, the apoptosis rate in both cell lines cotransfected with miR-18a and neogenin 30 -UTR was significantly decreased compared with that in the miR-18aþneogenin group (Po0.05; Fig. S3D).
Discussion In this study, we have found that the miR-18a expression was significantly increased in glioblastoma tissues and cell lines, while neogenin expression was clearly down-regulated. Furthermore, the expression levels of miR-18a and neogenin were directly and inversely (respectively) correlated to the pathological grade of glioma. MiR-18a negatively regulated the mRNA and protein expression of neogenin by binding to a specific site within the 30 -UTR of neogenin. Inhibition of miR-18a could remarkably alter the phenotype of human glioblastoma cells by inhibiting cellular proliferation, migration and invasion, inducing cell cycle arrest in G0/G1 phase, as well as promoting cellular apoptosis by upregulating the expression of neogenin. This is the first report, to date, outlining the phenotypic effects of miR-18a down-regulation in glioblastoma.
In recent years, increasing attention has been paid on the role of miRNAs in tumor development. MiR-18a, as an important member of miR-17-92 family, has shown various effects on different tumors. Firstly, as an oncogenic factor, miR-18a can inhibit the repair of damaged DNA by down-regulating the expression of ataxia telangiectasia mutated (ATM) in breast cancer cells [28]. MiR-18a in plasma also served as a novel potential marker in clinical screening or diagnosis for hepatitis B virus-related hepatocellular carcinoma, pancreatic cancer and esophageal squamous cell carcinoma [29–31]. Conversely, it was reported that miR-18a could act as a tumor suppressor as to inhibit the proliferation of T24 bladder cancer cells by down-regulating the expression of Dicer directly [32]. Therefore, the present research suggested that miR-18a could regulate various biological functions in different tumors by controlling target genes. In order to clarify the effects of miR-18a on glioblastoma, we first detected the expression of miR18a in collected clinical specimens and U87 and U251 human glioblastoma cell lines. The results showed that miR-18a expression was significantly up-regulated in glioblastoma tissues and cell lines compared with the human brain tissues and NHA. Furthermore, the expression level was increased with the rising pathological grades of glioblastoma, indicating that miR-18a might be an oncogene in glioblastoma. Our findings were consistent with previous studies, for example, Ernst reported that miR-18a expression in glioblastoma was up-regulated compared with normal brain tissues [33]. However, the underlying molecular mechanism relating the effects of miR-18a on glioblastoma still remains unclear. We attempted to predict the potential target genes of miR-18a in glioblastoma through applying the bioinformatics analysis method (TargetScan and Miranda), then we found neogenin, a theoretical target gene of miR-18a with the specific binding site in the 30 -UTR sequence.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945
E X PE R IM EN TA L C ELL R E S EA RC H
946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005
] (]]]]) ]]]–]]]
9
Fig. 8 – MiR-18a inhibitor promoted apoptosis in glioblastoma cells. Apoptosis of U87 and U251 cells was monitored by flow cytometry. Early apoptotic cells are Annexin Vþ/PI , late apoptotic cells are Annexin Vþ/PIþ, necrotic cells are Annexin V /PIþ and healthy cells are Annexin V /PI . The representative experiment of three groups was shown, and we found that the percentage of apoptotic cells in miR-18a inhibitor group was significantly increased compared with its negative control group (Po0.05). And there was no obvious difference between untransfected group and miR-18a inhibitor NC group. Data were presented as mean7SD of three independent experiments. *Po0.05, vs. the miR-18a inhibitor NC group.
Neogenin, highly similar to DCC in structure and function, plays an important role in the development of the central nervous system and has significant regulatory effects on a wide variety of biological activities such as formation of vessels and tissues as well as maintaining homeostasis after birth [34–36]. Recently neogenin was considered as a tumor suppressor in certain malignant tumors, such as lung adenocarcinomas [24], breast cancer [26], colon cancer [37] and prostate cancer [38] as the down-regulation of neogenin in these tumors could accelerate the tumor progression. Neogenin also acted as a tumor suppressor in glioblastoma because the down-regulation of its expression not only accelerated the tumorigenesis, but also influenced the migration and apoptosis of glioblastoma cells [27]. Nevertheless, the molecular mechanism underlying this process is still poorly understood. To clarify whether miR-18a could regulate the expression of neogenin and thereby influence the biological behavior of glioblastoma, we detected the neogenin expression level in glioblastoma. Results showed that neogenin expression was significantly down-regulated in glioblastoma tissues compared with the homologous surrounding non-neoplastic tissues, and the expression level was decreased with the rising pathological grades of glioblastoma. Wu et al. [27] reported that the distribution and expression of neogenin were narrower and lower in gliomas than their homologous surrounding areas, which was in accordance with our present work. Subsequently, we found that neogenin expression could be significantly up-regulated in the U87 and U251 cells by transfecting exogenous miR-18a inhibitor. Luciferase reporter assay also confirmed that miR-18a could target on the 30 -UTR of neogenin. Therefore, our results revealed the negative regulatory effects of miR-18a on neogenin expression in glioblastoma for the first time.
In our study, we also observed the inhibition of cellular proliferation, migration and invasion, induction of cell cycle arrest in the G0/G1 phase, as well as promotion of apoptosis in U87 and U251 cells by transfecting exogenous miR-18a inhibitor. In order to further understand the potential mechanism during the process, we co-transfected U87 and U251 cells with miR-18a expression vector and neogenin vectors with or without 30 -UTR. Results demonstrated that the effects of miR-18a on biological behavior of glioblastoma cells were related to the negative regulation of neogenin. We found that the expression level of neogenin was significantly down-regulated and cellular proliferation, migration and invasion were significantly recovered by co-transfection of miR-18a and neogenin with 30 -UTR. These data further validated the hypothesis that the changes of biological behavior in glioblastoma cells were due to the modulatory effects of miR-18a on neogenin. Therefore, inhibition of miR-18a could significantly suppress cell proliferation, migration and invasion and promote cellular apoptosis in glioblastoma cells by upregulating the expression of neogenin. Similarly, inhibition of miR-18a expression was reported to suppress these biological processes in other tumors as well, for example, inhibition of miR18a in neuroblastoma cells could lead to severe growth retardation by influencing the cell cycle [39]. Furthermore, recent studies also confirmed that the inhibition of other miRNAs could suppress the biological behavior of glioblastoma cells. Guessous et al. [40] found that inhibition of miR-10b expression significantly suppressed the cell proliferation, migration and invasion in glioblastoma cells and stem cells. Zhang et al. [41] found that inhibition of miR-106b expression could result in cell cycle arrest and suppress cell proliferation by up-regulating the expressions of p21 and RBL2 in U251 and LN229 glioblastoma cells. Taken these results
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065
10
1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125
E XP E RI ME N TAL CE L L R ES E ARC H
together, it is reasonable to infer that inhibiting the corresponding carcinogenic miRNAs might bring another therapeutic option for the treatment of malignant brain tumors. However, it is notable that even the same miRNA may produce the contrary effects in different tumors. For example, inhibition of miR-155 expression in glioblastoma U87 cells could significantly suppress the migration and invasion abilities and promote cellular apoptosis [42], whereas it could promote cell proliferation in human melanoma cells by up-regulating the expression of human SKI [43]. Therefore, how to inhibit the expression of correct target miRNAs precisely and specifically requires further investigation. In conclusion, this study revealed, for the first time, that inhibition of miR-18a expression could significantly suppress cell proliferation, migration and invasion, induce cell cycle arrest in G0/G1 phase and promote the apoptosis in glioblastoma U87 and U251 cells. The potential mechanism might be related to the increased levels of miR-18a-targeted neogenin. Therefore, as a potential novel target of glioblastoma, inhibition of miR-18a may provide a new therapeutic strategy for human glioblastoma treatment.
Acknowledgments Q2 This work is supported by grants from the Natural Science Founda-
tion of China (81172197, 81072056, 81171131, 81272564, and 81272795), the special fund for the Scientific Research of Doctordegree Subjects in Colleges and Universities (20102104110009), the Liaoning Science and Technology Plan Projects (No. 2011225020), the Shenyang Science and Technology Plan Projects (Nos. F12-277-1Q3 05, F13-316-1-16, and F13-220-9-15), and the Outstanding Scientific Fund of Shengjing Hospital.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2014.03. 009.
references [1] H. Ohgaki, Epidemiology of brain tumors, Methods Mol. Biol. 472 (2009) 323–342. [2] G. Li, Z.G. Zhang, Y.Y. Tu, T.B. Jin, H.J. Liang, Correlation of microrna-372 upregulation with poor prognosis in human glioma, Diagn. Pathol. 8 (2013) 1. [3] A. Stark, J. Brennecke, N. Bushati, R.B. Russell, S.M. Cohen, Animal microRNAs confer robustness to gene expression and have a significant impact on 30 UTR evolution, Cell 123 (2005) 1133–1146. [4] Y. Tian, Y. Nan, L. Han, A. Zhang, G. Wang, Z. Jia, J. Hao, P. Pu, Y. Zhong, C. Kang, MicroRNA miR-451 downregulates the PI3K/AKT pathway through CAB39 in human glioma, Int. J. Oncol. 40 (2012) 1105–1112. [5] S. Qu, Y. Yao, C. Shang, Y. Xue, J. Ma, Z. Li, Y. Liu, MicroRNA-330 is an oncogenic factor in glioblastoma cells by regulating SH3GL2 gene, PLoS One 7 (2012) e46010. [6] J.A. Chan, A.M. Krichevsky, K.S. Kosik, MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells, Cancer Res. 65 (2005) 6029–6033.
] (]]]]) ]]]–]]]
[7] Z. Lu, M. Liu, V. Stribinskis, C.M. Klinge, K.S. Ramos, N.H. Colburn, Y. Li, MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene, Oncogene 27 (2008) 4373–4379. [8] J.K. Gillies, I.A. Lorimer, Regulation of p27Kip1 by miRNA 221/222 in glioblastoma, Cell Cycle 6 (2007) 2005–2009. [9] Z.M. Shi, J. Wang, Z. Yan, Y.P. You, C.Y. Li, X. Qian, Y. Yin, P. Zhao, Y.Y. Wang, X.F. Wang, M.N. Li, L.Z. Liu, N. Liu, B.H. Jiang, MiR-128 inhibits tumor growth and angiogenesis by targeting p70S6K1, PLoS One 7 (2012) e32709. [10] J. Silber, A. Jacobsen, T. Ozawa, G. Harinath, A. Pedraza, C. Sander, E.C. Holland, J.T. Huse, MiR-34a repression in proneural malignant gliomas upregulates expression of its target PDGFRA and promotes tumorigenesis, PLoS One 7 (2012) e33844. [11] S. Takakura, N. Mitsutake, M. Nakashima, H. Namba, V.A. Saenko, T.I. Rogounovitch, Y. Nakazawa, T. Hayashi, A. Ohtsuru, S. Yamashita, Oncogenic role of miR-17-92 cluster in anaplastic thyroid cancer cells, Cancer Sci. 99 (2008) 1147–1154. [12] B. Diosdado, M.A. van de Wiel, J.S. Terhaar Sive Droste, S. Mongera, C. Postma, W.J. Meijerink, B. Carvalho, G.A. Meijer, MiR17-92 cluster is associated with 13q gain and c-myc expression during colorectal adenoma to adenocarcinoma progression, Br. J. Cancer 101 (2009) 707–714. [13] M. Karsy, E. Arslan, F. Moy, Current progress on understanding microRNAs in glioblastoma multiform, Genes Cancer 3 (2012) 3–15. [14] W.H. Liu, S.H. Yeh, C.C. Lu, S.L. Yu, H.Y. Chen, C.Y. Lin, D.S. Chen, P.J. Chen, MicroRNA-18a prevents estrogen receptor-alpha expression, promoting proliferation of hepatocellular carcinoma cells, Gastroenterology 136 (2009) 683–693. [15] W. Wu, M. Takanashi, N. Borjigin, S.I. Ohno, K. Fujita, S. Hoshino, Y. Osaka, A. Tsuchida, M. Kuroda, MicroRNA-18a modulates STAT3 activity through negative regulation of PIAS3 during gastricadenocarcinogenesis, Br. J. Cancer 108 (2013) 653–661. [16] L. Turchi, D.N. Debruyne, F. Almairac, V. Virolle, M. Fareh, Y. Neirijnck, F. Burel-Vandenbos, P. Paquis, M.P. Junier, E. Van Obberghen-Schilling, H. Chneiweiss, T. Virolle, Tumorigenic potential of miR-18An in glioma initiating cells requires NOTCH-1 signaling, Stem Cells 31 (2013) 1252–1265. [17] J.L. Fox, M. Dews, A.J. Minn, A. Thomas-Tikhonenko, Targeting of TGFβsignature andits essential component CTGF by miR-18 correlates with improved survival in glioblastoma, RNA 19 (2013) 177–190. [18] N.H. Wilson, B. Key, Neogenin: one receptor, many functions, Int. J. Biochem. Cell Biol. 39 (2007) 874–878. [19] S.J. Cole, D. Bradford, H.M. Cooper, Neogenin: a multi-functional receptor regulating diverse developmental processes, Int. J. Biochem. Cell Biol. 39 (2007) 1569–1575. [20] M. De Vries, H.M. Cooper, Emerging roles for neogenin and its ligands in CNS development, J. Neurochem. 106 (2008) 1483–1492. [21] E.R. Fearon, K.R. Cho, J.M. Nigro, S.E. Kern, J.W. Simons, J.M. Ruppert, S.R. Hamilton, A.C. Preisinger, G. Thomas, K.W. Kinzler, B. Vogelstein, Identification of a chromosome 18q gene that is altered in colorectal cancers, Science 247 (1990) 49–56. [22] S. Rodrigues, O. De Wever, E. Bruyneel, R.J. Rooney, C. Gespach, Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis, Oncogene 26 (2007) 5615–5625. [23] K. Thiebault, L. Mazelin, L. Pays, F. Llambi, M.O. Joly, J.Y. Scoazec, J.C. Saurin, G. Romeo, P. Mehlen, The netrin-1 receptors UNC5H are putative tumor suppressors controlling cell death commitment, Proc. Natl. Acad. Sci. USA 100 (2003) 4173–4178. [24] P. Mehlen, C. Thibert, Dependence receptors: between life and death, Cell. Mol. Life Sci. 61 (2004) 1854–1866. [25] D. Berrar, B. Sturgeon, I. Bradbury, C.S. Downes, W. Dubitzky, Survival trees for analyzing clinical outcome in lung adenocarcinomas based on gene expression profiles: identification of neogenin and diacylglycerol kinase alpha expression as critical factors, J. Comput. Biol. 12 (2005) 534–544.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185
E X PE R IM EN TA L C ELL R E S EA RC H
1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220
[26] J.E. Lee, H.J. Kim, J.Y. Bae, S.W. Kim, J.S. Park, H.J. Shin, W. Han, S.W. Kim, K.S. Kang, D.Y. Noh, Neogenin expression may be inversely correlated to the tumorigenicity of human breast cancer, BMC Cancer 5 (2005) 154. [27] X. Wu, Y. Li, X. Wan, T.M. Kayira, R. Cao, X. Ju, X. Zhu, G. Zhao, Down-regulation of neogenin accelerated glioma progression through promoter methylation and its overexpression in SHG-44 induced apoptosis, PLoS One 7 (2012) e38074. [28] L. Song, C. Lin, Z. Wu, H. Gong, Y. Zeng, J. Wu, M. Li, J. Li, MiR-18a impairs DNA damage response through downregulation of ataxia telangiectasia mutated(ATM) kinase, PLoS One 6 (2011) e25454. [29] L. Li, Z. Guo, J. Wang, Y. Mao, Q. Gao, Serum miR-18a: a potential marker for hepatitis B virus-related hepatocellular carcinoma screening, Dig. Dis. Sci. 57 (2012) 2910–2916. [30] R. Morimura, S. Komatsu, D. Ichikawa, H. Takeshita, M. Tsujiura, H. Nagata, H. Konishi, A. Shiozaki, H. Ikoma, K. Okamoto, T. Ochiai, H. Taniguchi, E. Otsuji, Novel diagnostic value of circulating miR-18a in plasma of patients with pancreatic cancer, Br. J. Cancer 105 (2011) 1733–1740. [31] S. Hirajima, S. Komatsu, D. Ichikawa, H. Takeshita, H. Konishi, A. Shiozaki, R. Morimura, M. Tsujiura, H. Nagata, T. Kawaguchi, T. Arita, T. Kubota, H. Fujiwara, K. Okamoto, E. Otsuji, Clinical impact of circulating miR-18a in plasma of patients with oesophageal squamous cell carcinoma, Br. J. Cancer 108 (2013) 1822– 1829. [32] J. Tao, D. Wu, P. Li, B. Xu, Q. Lu, W. Zhang, MicroRNA-18a, a member of the oncogenic miR-17-92 cluster, targets Dicer and suppresses cell proliferation inbladder cancer T24 cells, Mol. Med. Rep. 5 (2012) 167–172. [33] A. Ernst, B. Campos, J. Meier, F. Devens, F. Liesenberg, M. Wolter, G. Reifenberger, C. Herold-Mende, P. Lichter, B. Radlwimmer, Derepression of CTGF via the miR-17-92 cluster upon differentiation of human glioblastoma spheroid cultures, Oncogene 29 (2010) 3411–3422. [34] Y. Han, Y. Shao, Z. Lin, Y.L. Qu, H. Wang, Y. Zhou, W. Chen, Y. Chen, W.L. Chen, F.R. Hu, W. Li, Z. Liu, Netrin-1 simultaneously suppresses corneal inflammation and neovascularization, Investig. Ophthalmol. Vis. Sci. 53 (2012) 1285–1295.
] (]]]]) ]]]–]]]
11
[35] T. Ito, M. Kagoshima, Y. Sasaki, C. Li, N. Udaka, T. Kitsukawa, H. Fujisawa, M. Taniguchi, T. Yagi, H. Kitamura, Y. Goshima, Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung, Mech. Dev. 97 (2000) 35–45. [36] C. Jayakumar, R. Mohamed, P.V. Ranganathan, G. Ramesh, Intracellular kinases mediate increased translation and secretion of netrin-1 from renal tubular epithelial cells, PLoS One 6 (2011) e26776. [37] V.S. Li, S.T. Yuen, T.L. Chan, H.H. Yan, W.L. Law, B.H. Yeung, A.S. Chan, W.Y. Tsui, S. So, X. Chen, S.Y. Leung, Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology 137 (2009) 176–187. [38] A. Latil, L. Chene, B. Cochant-Priollet, P. Mangin, G. Fournier, P. Berthon, O. Cussenot, Quantification of expression of netrins, slits and their receptors in human prostate tumors, Int. J. Cancer 103 (2003) 306–315. [39] J. Lovén, N. Zinin, T. Wahlström, I. Müller, P. Brodin, E. Fredlund, U. Ribacke, A. Pivarcsi, S. Påhlman, M. Henriksson, MYCNregulated microRNAs repress estrogen receptor-alpha (ESR1) expression and neuronal differentiation inhuman neuroblastoma, Proc. Natl. Acad. Sci. USA 107 (2010) 1553–1558. [40] F. Guessous, M. Alvarado-Velez, L. Marcinkiewicz, Y. Zhang, J. Kim, S. Heister, B. Kefas, J. Godlewski, D. Schiff, B. Purow, R. Abounader, Oncogenic effects of miR-10b in glioblastoma stem cells, J. Neurooncol. 112 (2013) 153–163. [41] A. Zhang, J. Hao, K. Wang, Q. Huang, K. Yu, C. Kang, G. Wang, Z. Jia, L. Han, P. Pu, Down-regulation of miR-106b suppresses the growth of human glioma cells, J. Neurooncol. 112 (2013) 179–189. [42] N. Ling, J. Gu, Z. Lei, M. Li, J. Zhao, H.T. Zhang, X. Li, microRNA-155 regulates cell proliferation and invasion by targeting FOXO3a in glioma, Oncol. Rep. 30 (2013) 2111–2118. [43] L. Levati, E. Pagani, S. Romani, D. Castiglia, E. Piccinni, C. Covaciu, P. Caporaso, S. Bondanza, F.R. Antonetti, E. Bonmassar, F. Martelli, E. Alvino, S. D'Atri, MicroRNA-155 targets the SKI gene in human melanoma cell lines, Pigment Cell Melanoma Res. 24 (2011) 538–550.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93
] (]]]]) ]]]–]]]
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/yexcr
Research Article
MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin Q1
Yichen Songa, Ping Wangb,c, Wei Zhaob,c, Yilong Yaoa, Xiaobai Liud, Jun Mab,c, Yixue Xueb,c, Yunhui Liua,n a
Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang 110004, People's Republic of China Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang 110001, People's Republic of China c Institute of Pathology and Pathophysiology, China Medical University, Shenyang 110001, People's Republic of China d The 96th Class, 7-year Program, China Medical University, Shenyang, Liaoning Province 110001, People's Republic of China b
article information
abstract
Article Chronology:
MiR-17-92 cluster has recently been reported as an oncogene in some tumors. However, the
Received 5 November 2013
association of miR-18a, an important member of this cluster, with glioblastoma remains
Received in revised form
unknown. Therefore, this study aims to investigate the expression of miR-18a in glioblastoma
5 March 2014
and its role in biological behavior of U87 and U251 human glioblastoma cell lines. Quantitative
Accepted 10 March 2014
RT-PCR results showed that miR-18a was highly expressed in glioblastoma tissues and U87 and U251 cell lines compared with that in human brain tissues and primary normal human
Keywords:
astrocytes, and the expression levels were increased along with the rising pathological grades
Glioblastoma
of glioblastoma. Neogenin was identified as the target gene of miR-18a by dual-luciferase reporter
MiR-18a
assays. RT-PCR and western blot results showed that its expression levels were decreased along
Neogenin
with the rising pathological grades of glioblastoma. Inhibition of miR-18a expression was
Proliferation
established by transfecting exogenous miR-18a inhibitor into U87 and U251 cells, and its effects
Migration
on the biological behavior of glioblastoma cells were studied using CCK-8 assay, transwell assay
Invasion
and flow cytometry. Inhibition of miR-18a expression in U87 and U251 cells significantly upregulated neogenin, and dramatically suppressed the abilities of cell proliferation, migration and invasion, induced cell cycle arrest and promoted cellular apoptosis. Collectively, these results suggest that miR-18a may regulate biological behavior of human glioblastoma cells by targeting neogenin, and miR-18a can serve as a potential target in the treatment of glioblastoma. & 2014 Published by Elsevier Inc.
Introduction Glioblastoma is one of the most common primary malignant tumors in the adult central nervous system. Although treatment
strategies of surgery, radiation and chemotherapy have been well developed in recent years, patients with glioblastoma show no significantly improved prognosis and the median survival is reported to be only 14 months [1,2]. Therefore, glioblastoma
n
Corresponding author. E-mail addresses: [email protected] (Y. Song), [email protected] (P. Wang), [email protected] (W. Zhao), [email protected] (Y. Yao), [email protected] (X. Liu), [email protected] (J. Ma), [email protected] (Y. Xue), [email protected] (Y. Liu). http://dx.doi.org/10.1016/j.yexcr.2014.03.009 0014-4827/& 2014 Published by Elsevier Inc.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
94 95 96 97 98 99 100 101 102 103 104 105
2
106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165
E XP E RI ME N TAL CE L L R ES E ARC H
remains one of the most intractable diseases in the field of neurosurgery. MicroRNAs (miRNAs) are a class of about 20–22 bp small noncoding RNAs. They act either as oncogenes or tumor suppressors, and regulate gene expression post-transcriptionally by binding with 30 -UTR which causes either degradation or repression of target mRNAs [3,4]. Many miRNAs were shown to be abnormally expressed in glioblastoma, and were involved in the development of glioblastoma [4,5]. It was reported that miR-21 was strongly overexpressed in human glioblastoma and could induce the deterioration of glioblastoma by blocking the expression of apoptosis-related genes [6,7]. Recently, it has been reported that miR-221/222 overexpression promoted the aggressive growth of human glioblastoma by regulating the cell cycle related factor p27 [8]. In addition, miR-128 and miR-34a also acted as tumor suppressors in glioblastoma [9,10]. Therefore, different miRNAs may produce different biological effects on glioblastoma. MiR-17-92 cluster, recognized as an oncogene family, was overexpressed in anaplastic thyroid cancer and colorectal tumors and might be involved in controlling the corresponding target genes to regulate the development of these tumors [11,12]. It has been reported that miR-17-92 might play a significant role in the development of glioblastoma since its expression was up-regulated in the tumor [13]. MiR-18a, an important member of miR-17-92 cluster, plays a carcinogenic role in some tumors. For example, it promoted the proliferation of hepatoma cells by inhibiting ERalpha expression, and acted as oncogene during gastric adenocarcinogenesis by negatively regulating the expression of protein inhibitor against activated signal transducer and activator of transcription 3 (STAT3) [14,15]. At present, the effects of miR-18a on glioblastoma were still rarely reported. Turchi et al. [16] demonstrated the contribution of miR-18an to the promotion of glioma initiating cells self-renewal and tumorigenicity. On the contrary, Fox et al. [17] found that miR-18a was correlated with improved survival in glioblastoma patients by regulating transforming growth factor-β signal transduction and connective tissue growth factor expression. Therefore, the function and molecular mechanism of miR-18a in human glioblastoma are still unclear. Neogenin, a homolog of Deleted in Colorectal Cancer (DCC), displays wide distribution and lifelong stable expression in the central nervous system as a dependent receptor of neurogenesisrelated proteins like netrin, or members of the repulsive guidance molecule family [18]. Similar to DCC, neogenin binds with its ligands to play various physiological roles in normal tissues, including axon guidance, cell migration, tissue morphogenesis and differentiation, angiogenesis, signal transduction and cellular apoptosis [18–20]. DCC and its homolog neogenin have been considered as tumor suppressors in a variety of tumors since loss of DCC expression was discovered in colorectal cancers [21–24]. Recent studies showed that neogenin expression was downregulated in lung adenocarcinomas and breast cancer, and the low expressed neogenin could accelerate the tumor progression [25,26]. In the central nervous system, neogenin acted as a tumor suppressor and was lowly expressed in glioblastoma compared with control brain tissues [27]. As previously mentioned, miR-18a could affect the tumor development by regulating the expression of related genes; however, whether miR-18a has regulatory effects on neogenin in glioblastoma remains unclear. Under such background, the major aim of this study was to investigate the regulatory effects of miR-18a on neogenin and biological behavior of glioblastoma cells.
] (]]]]) ]]]–]]]
Material and methods Clinical specimens Glioblastoma specimens and their homologous surrounding nonneoplastic tissues were obtained from the Department of Neurosurgery, Shengjing Hospital of China Medical University, China, from September 2012 to July 2013. Fresh human brain lobectomy specimens obtained from surgeries of malignant intracranial hypertension such as severe intracerebral hemorrhage or craniocerebral injury were used as negative control. All specimens were immediately frozen and preserved in liquid nitrogen following surgical resection. Glioma specimens were classified into four grades by two experienced clinical pathologists according to the 2007 WHO classification of tumors in the central nervous system, then they were divided into two groups: low-grade glioma group (WHO I–II, n¼ 3) and high-grade glioma group (WHO III–IV, n¼3). The research methods in our study were approved by the Institutional Review Board at the Shengjing Hospital of China Medical University, China. All participants provided their written informed consent, and the hospital ethical committee approved the experiments.
Reagents and cell culture High-glucose Dulbecco's modified Eagle medium (DMEM), Dulbecco's modified Eagle medium/F12 mixed medium and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). Trizol, Opti-MEMs I and Lipofectamine2000 transfection reagents were purchased from Invitrogen (Carlsbad, CA, USA). MicroRNA-18a inhibitor and its negative control, pmirGLOneogenin 30 -UTR and pmirGLO-neogenin 30 -UTR-MUT dualluciferase miRNA target expression vectors, human neogenin with or without its 30 -UTR expression vectors pGCMV-neogenin-30 -UTR and pGCMV-neogenin were synthesized from GenePharma (Shanghai, China). pRI-CMV/GFP-hsa-miR-18a and its negative control were synthesized from Inovogen (Beijing, China). All other reagents and chemicals were purchased from Sigma-Aldrich (Shanghai, China). Human glioblastoma cell lines U87 and U251, and human embryonic kidney cell line HEK-293 were obtained from the Chinese Academy of Medical Sciences (Beijing, China). U87 and HEK-293 cells were cultured in high glucose DMEM medium supplemented with 10% FBS, and U251 cells were cultured in DMEM/F12 medium supplemented with 10% FBS. All cells were maintained in a humidified incubator at 37 1C with 5% CO2. Primary normal human astrocytes (NHA) were purchased from the Sciencell Research Laboratories (Carlsbad, CA, USA) and cultured under the instructed condition by the manufacturer.
Cell transfection Glioma cells at approximately 50–70% confluence were transfected using Opti-MEMs I and Lipofectamine2000 reagents (Invitrogen, CA, USA) after 24 h of culture. Transfection complexes and methods were prepared according to the manufacturer's instructions. MiR-18a inhibitor sequence: 50 -CUA UCU GCA CUA GAU GCA CCU UA-30 and miR-18a inhibitor negative control (NC) sequence: 50 -CAG UAC UUU UGU GUA GUA CAA-30 . After 6 h of transfection, the transfection medium was replaced with
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225
E X PE R IM EN TA L C ELL R E S EA RC H
226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285
high-glucose DMEM or DMEM/F12 medium with 10% FBS, and the cells were incubated for additional 24–96 h, then the transfected cells were harvested for further studies. U87 and U251 cells transfected with miR-18a inhibitor or its negative control were divided into three groups according to the experimental requirement: untransfected group, miR-18a inhibitor NC group and miR18a inhibitor group. Meanwhile, U87 and U251 cells cotransfected with pRI-CMV/GFP-hsa-miR-18a or its negative control and pGCMV-neogenin, and pGCMV-neogenin-30 -UTR or their negative control were divided into four groups: miR-18a-conþempty vector (EV) group; miR-18aþEV group; miR18aþneogenin group; miR-18aþneogenin 30 -UTR group.
Bioinformatics prediction and luciferase reporter assay The common targets of miR-18a predicted by computer-aided algorithms were obtained from multiple target prediction programs: Targetscan and Miranda (http://www.targetscan.org/ and http:// www.microrna.org/). HEK-293 cells were seeded in 96-well plates for 24 h, then cells at 50–70% confluence were co-transfected with wild-type or mutated pmirGLO-neogenin 30 -UTR reporter plasmid and pRI-CMV/GFP-hsa-miR-18a or pRI-CMV/GFP-hsa-miR-18a-con. HEK-293 cells were divided into five groups: pmirGLO group (blank); miR-18aþpmirGLO-neogenin 30 -UTR group; miR-18aþ pmirGLO-neogenin 30 -UTR-MUT group; miR-18a-conþpmirGLO-neo genin 30 -UTR group; miR-18a-conþpmirGLO-neogenin 30 -UTR-MUT group. Luciferase activity assays were performed at 48 h after transfection using the dual-luciferase reporter assay system (Pro mega, Madison, WI, USA). Renilla luciferase activity was used as an internal control.
RNA isolation, RT-PCR and quantitative RT-PCR (qRT-PCR) Total RNA of each group was extracted from the tissues and NHA, U87, and U251 cells using Trizol according to the manufacturer's instructions. After extraction, total RNA was divided into two parts. One part was used for qRT-PCR analysis of miR-18a, using TaqMans MicroRNA RT kit (Applied Biosystems, Carlsbad, CA, USA) for reverse transcription, then using TaqMans Universal Master Mix II (Applied Biosystems) and ABI 7500 Fast Real-Time PCR System for qRT-PCR analysis. The reaction conditions: 50 1C for 2 min, 95 1C for 10 min, and 40 cycles of 95 1C for 15 s, and 60 1C for 1 min. The expression level of miR-18a was normalized with reference to expression level of U6, and fold changes were calculated by relative quantification (2 ΔΔCt). The primers of miR18a (Hs01932578_s1) and U6 (001973) were synthesized from the Applied Biosystems. The other part of total RNA was used to detect mRNA level of neogenin, according to RNA PCR Kit (AMV) Ver.3.0 (TaKaRa, Dalian, Liaoning Province China), RNA was reversed into cDNA, then amplified by PCR using the following primers: neogenin forward: 50 -GAC TCC CGA TAC TAC ACC GT-30 , reverse: 50 -AAT TGG ACA GCT TCA GAC ATG-30 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward: 50 -GGT GAA GGT CGG AGT CAA CG-30 , reverse 50 -CCA TGT AGT TGA GGT CAA TGA AG-30 . The reaction conditions: 35 cycles of 94 1C denaturation for 30 s, 55 1C annealing for 30 s, and 72 1C extension for 1 min. The products were electrophoresed using 1% agarose gel and stained with GeneFinder. The relative neogenin mRNA level was normalized to that of GAPDH mRNA level using Quality One analysis software (Bio-Rad Laboratories, Hercules, CA,
] (]]]]) ]]]–]]]
3
USA). The primers for RT-PCR were synthesized from Sangon Biotech (Shanghai, China).
Western blot analysis Total protein from tissues and cells was extracted in RIPA buffer (Beyotime, Haimen, Jiangsu Province, China) supplemented with 1% phenylmethyl sulfonylfluoride. The samples were centrifuged at 12,000g, 4 1C for 20 min and the protein concentration was determined by the BCA method (Beyotime). Equal amounts of protein samples were separated using 10% sodium dodecyl sulfate polyacrylamide gel electropheresis and then electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore, Shanghai, China). The membranes were blocked in blocking buffer (5% non-fat milk dissolved in Tris–buffered saline–Tween) overnight at 4 1C. The blots were then respectively incubated with rabbit polyclonal anti-neogenin (sc-15337) antibody (1:400; Santa Cruz Biotechnology, Dallas, Texas, USA) and mouse monoclonal anti-GAPDH (sc-47724) antibody (1:5 000; Santa Cruz Biotechnology) overnight at 4 1C. Then the blots were incubated with corresponding secondary antibody at room temperature for 2 h after four washes for 30 min. Finally, protein bands were visualized by ECL (Santa Cruz Biotechnology) and detected by ECL Detection Systems (Thermo Scientific, Beijing, China). The relative integrated density values were calculated based on GAPDH protein as an internal control.
Cell proliferation assay U87 and U251 cells were seeded in 96-well plates at the density of 2000 cells/well, and cell inhibitory rate was assayed using the Cell Counting Kit-8 (Beyotime) at 24, 48, 72, and 96 h after transfection. 10 μL CCK-8 solution was added into each well and cells were incubated for another 1.5 h in a humidified incubator. Optical density value was measured at 450 nm. Five replicate wells were set up in each group and repeated three independent experiments were performed.
Cell migration and invasion assay Migration and invasion of U87 and U251 cells in vitro were assayed using a Transwell chamber (Costar, Corning, NY, USA) with a polycarbonic membrane (6.5 mm in diameter, 8 μm pore size). In the migration assay, cellular medium was replaced with serum-free medium after 24 h of transfection and cells were incubated for another 24 h. Then cells were trypsinized and suspended into single cells with serum-free medium at the density of 5 105 cells/mL. 100 μL of the cell suspension was added to the upper chamber, and 600 mL of high-glucose DMEM or DMEM/F12 medium supplemented with 10% FBS was added to the lower chamber. Cells were incubated for 24 h at 37 1C, then non-migrating cells on the top surface of membrane were removed with cotton swabs. Cells that migrated to the lower surface of the membrane were then fixed with methanol and stained with 20% Giemsa solution for 30 min at 37 1C and washed twice with phosphate buffer saline (PBS). Then stained cells were observed under an inverted microscope (400 ) to count the cell number within five randomly chosen fields and the average number was calculated. In the invasion assay, the transwell membrane was coated with 80 mL of Matrigel solution
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345
4
346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405
E XP E RI ME N TAL CE L L R ES E ARC H
] (]]]]) ]]]–]]]
(500 ng/mL; BD, Franklin Lakes, NJ, USA) and incubated at 37 1C for 4 h, the remaining steps were similar to the migration assay.
Cell cycle analysis Cell cycle assay was performed at 48 h after transfection. Cells in each group were trypsinized and resuspended in ice-cold PBS and gently pelleted by centrifugation at 2000 rpm, 4 1C for 5 min, then the cell density was adjusted to 1 106 cells/mL with ice-cold PBS. After being centrifuged at 2000 rpm, 4 1C for 5 min, cells were fixed and permeabilized in 70% ethanol overnight at 20 1C. Fixed cells were washed with PBS and incubated with 100 μL RNase A solution in water bath at 37 1C for 30 min according to the instruction of Cell Cycle Assay (KeyGEN, Nanjing, Jiangsu Province, China), then 400 μL of propidium iodide (PI) staining solution was added and the system was kept in dark at 4 1C for 30 min. Finally, cells were analyzed for DNA content on BD FACSCalibur (BD Biosciences, San Jose, CA, USA).
Apoptosis detection The effect of miR-18a expression on cellular apoptosis was assessed by Annexin V-FITC/ PI double staining kit (Beyotime). At 48 h after transfection, cells were collected and stained with Annexin V-FITC and PI according to the manufacturer's instruction, then they were analyzed on BD FACSCalibur.
Statistical analysis All the above experiments were independently repeated three times. All results were expressed as mean7SD, and all data were analyzed using SPSS 13.0 software (SPSS, Chicago, IL, USA). Group comparisons were analyzed with one-way analysis of variance, with Po0.05 as statistically significant difference.
Results Up-regulation of miR-18a expression in glioblastoma tissues and cell lines The expression levels of miR-18a mRNA in control brain, lowgrade glioma, high-grade glioma tissues and NHA, U87, and U251 cells were analyzed by qRT-PCR. As shown in Fig. 1, miR-18a mRNA expression was significantly up-regulated in glioblastoma tissues and two glioblastoma cell lines compared with the average expression levels in control brain tissues and NHA (Po0.05). Furthermore, the expression levels were elevated with the rising pathological grades of glioblastoma.
Down-regulation of neogenin expression in glioblastoma tissues and cell lines The neogenin mRNA and protein expression levels in control brain tissues, glioblastoma tissues and NHA, U87, and U251 cells were analyzed using RT-PCR and western blot assays, respecticvely. The RT-PCR results (Fig. 2A) showed that, neogenin mRNA expression was significantly down-regulated in glioblastoma tissues and two glioblastoma cell lines compared with that in homologous surrounding non-neoplastic tissues and NHA (Po0.05). Furthermore,
Fig. 1 – MiR-18a was overexpressed in human glioblastoma tissues and cell lines. Expression level of miR-18a was assessed in control brain tissues, low-grade glioma tissues, high-grade glioma tissues and NHA, U87, and U251 cells by qRT-PCR. U6 was used as an internal control. Data were presented as mean7SD from three independent experiments. *Po0.05, vs. the control group and #Po0.05, vs. NHA.
the expression level was also lower in high-grade glioma tissues than in low-grade glioma tissues. The western blot analysis results (Fig. 2B) were similar to RT-PCR, while neogenin protein expression was negative in U87 and U251 cells.
MiR-18a inhibitor significantly up-regulated neogenin expression in glioblastoma cells The expression of miR-18a and neogenin in U87 and U251 cells transfected with exogenous miR-18a inhibitor was analyzed at 24, 48, 72, and 96 h after transfection. The qRT-PCR results showed that the expression level of miR-18a was lowest at 48 h after transfection (Fig. S1A), likewise, the expression levels of neogenin mRNA and protein were highest at 48 h after transfection (Fig. S1B). Therefore, the regulatory effects of miR-18a on neogenin expression in glioblastoma cells were detected at 48 h after transfection in the following experiments. After U87 and U251 cells were transfected for 48 h, the miR-18a expression was significantly decreased compared with that of miR-18a inhibitor NC group, while there was no obvious difference between untransfected group and miR-18a inhibitor NC group (Fig. 3A). Meanwhile, as shown in Figs. 3B and C, the mRNA and protein expressions of neogenin in U87 and U251 cells were significantly increased in miR-18a inhibitor group compared with that of miR18a inhibitor NC group; however, there was no apparent difference between untransfected group and miR-18a inhibitor NC group. These results suggested that miR-18a could negatively regulate neogenin mRNA and protein expressions in U87 and U251 cells.
MiR-18a targeted the neogenin 30 -UTR The target genes of miR-18a were identified through bioinformatics analyses available on the miRNA database (http://www.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465
E X PE R IM EN TA L C ELL R E S EA RC H
466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525
] (]]]]) ]]]–]]]
5
Fig. 2 – Neogenin expression was significantly down-regulated in human glioblastoma tissues and cell lines. (A) The neogenin mRNA level was decreased in glioma tissues (T) compared with the homologous surrounding non-neoplastic tissues (S) (Po0.05). The neogenin mRNA level in U87 and U251 cells (C) was also decreased compared with that in NHA (C) (Po0.05). The expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. (B) The results of western blot analysis were similar to RT-PCR, while the neogenin in U87 and U251 cells was loss of expression. GAPDH was used as an internal control. Data was presented as mean7SD from three independent experiments. *Po0.05, vs. the homologous surrounding nonneoplastic tissues (S) and #Po0.05, vs. NHA (C).
Fig. 3 – MiR-18a negatively regulated the expression of neogenin in glioblastoma cells. (A) The expression level of miR-18a was assessed in transfected U87 and U251 cells. The results of qRT-PCR showed that miR-18a expression was decreased in miR-18a inhibitor group compared with its negative control group (Po0.05). U6 was used as an internal control. (B) The expression level of neogenin in U87 and U251 cells transfected with exogenous miR-18a inhibitor was changed. The RT-PCR results showed that the neogenin mRNA level was significantly up-regulated in miR-18a inhibitor group compared with its negative control group (Po0.05). (C) The western blot analysis results were similar to RT-PCR. Accompanying graphs show densitometry analysis of neogenin expression. Data was presented as mean7SD from three independent experiments. *Po0.05, vs. the miR-18a inhibitor NC group. Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585
6
586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645
E XP E RI ME N TAL CE L L R ES E ARC H
targetscan.org/ and http://www.microrna.org/). We found that the mRNA of neogenin was theoretically the target gene of miR-18a and its sequence has been maintained as highly bio-conservative in different species, such as the Homo sapiens, the Pan troglodytes , the Macaca mulatta etc. (Fig. 4A). So we speculated that miR-18a could target the 30 -UTR of neogenin. The wild- or mutated-type neogenin 30 -UTR was cloned into pmirGLO dual-luciferase miRNA target expression vector (Fig. 4B), and these constructs were validated by sequencing. Then the miR-18a, miR-18a-con and neogenin wild or mutated type luciferase reporter plasmids were co-transfected in HEK293 cells. Dual-Luciferases reporter assay system showed that, overex pressed miR-18a significantly decreased luciferase activity of wild-type pmirGLO-neogenin 30 -UTR compared with the miR-18a-con group, and miR-18a did not affect mutated pmirGLO-neogenin 30 -UTR-MUT luciferase activity (Fig. 4C). Collectively, these results indicated that neogenin might be the target of miR-18a.
MiR-18a inhibitor decreased the proliferation of glioblastoma cells The ability of cell proliferation was analyzed at 24, 48, 72, and 96 h after transfection (Fig. 5). The results demonstrated that the U87 and U251 cells transfected with miR-18a inhibitor exhibited a significant decrease of cell proliferation compared with miR-18a inhibitor NC group (Po0.05), while there was no apparent difference between untransfected group and miR-18a inhibitor NC group (P40.05; Fig. 5A). Furthermore, the inhibition rate of cell proliferation in miR-18a inhibitor group reached the maximum point at 48 h after transfection (Fig. 5B). In order to further validate the potential mechanism of biological behavior changes of glioblastoma cells related to the negative regulatory effects of miR-18a on neogenin, a rescue experiment was performed by co-transfecting miR-18a expression vector and pGCMV-neogenin or pGCMV-neogenin-30 -UTR in U87 and U251 cells. As shown in Fig. S2, the mRNA and protein expression levels of neogenin were
] (]]]]) ]]]–]]]
significantly down-regulated in miR-18aþneogenin 30 -UTR group compared with the miR-18aþneogenin group (Po0.05). And the results of the CCK-8 assay showed that the down-regulation of neogenin expression significantly promoted the proliferation of U87 and U251 cells (Fig. S3A).
MiR-18a inhibitor inhibited the migration and invasion of glioblastoma cells The migration and invasion of the U87 and U251 cells were examined at 48 h after transfection (Fig. 6). The results showed that the migration and invasion ability of U87 and U251 cells transfected with miR-18a inhibitor was significantly decreased compared with miR-18a inhibitor NC group (Po0.05), while there was no noticeable difference between the untransfected group and miR-18a inhibitor NC group (P40.05). The results of the rescue experiment showed that migration and invasion capacities of miR-18aþneogenin 30 -UTR group was obviously higher than that in miR-18aþneogenin group (Po0.05; Fig. S3B).
MiR-18a inhibitor induced G0/G1 arrest in glioblastoma cells The cell cycle in U87 and U251 cells was analyzed by flow cytometry at 48 h after transfection (Fig. 7). The results showed that the percentage of cells increased in G0/G1 phase and decreased in S and G2/M phase significantly in U87 and U251 cells transfected with miR-18a inhibitor compared with the miR18a inhibitor NC group (Po0.05), while there was no apparent difference between untransfected group and miR-18a inhibitor NC group. In the rescue experiment, the miR-18aþneogenin 30 -UTR group showed a lower percentage of cells in G0/G1 phase and a higher percentage of cells in S and G2/M phase compared with that in miR-18aþneogenin group (Po0.05; Fig. S3C).
Fig. 4 – MiR-18a negatively regulated neogenin expression through binding with neogenin 30 -UTR. (A) Schematic diagram of putative miR-18a binding site in the 30 -UTR of neogenin in human. The nucleotide sequence illustrates the predicted base-pairing between miR-18a and neogenin 30 -UTR. (B) The corresponding mutant nucleotides of the neogenin 30 -UTR was labeled in bold below. (C) MiR-18a down-regulated luciferase activities controlled by wild-type neogenin 30 -UTR, but did not affect luciferase activities controlled by mutant-type neogenin 30 -UTR, moreover, miR-18a-con did not affect luciferase activities controlled by wild-type or mutant-type neogenin 30 -UTR. Relative luciferase activities were calculated as the ratio of firefly/renilla activities in the cells and normalized to the pmirGLO group. Data were presented as mean7SD from three independent experiments. *Po 0.05, vs. the miR-18aþpmirGLO-neogenin 30 -UTR group. Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705
E X PE R IM EN TA L C ELL R E S EA RC H
706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765
] (]]]]) ]]]–]]]
7
Fig. 5 – MiR-18a inhibitor reduced cell proliferation in glioblastoma cells. CCK-8 assay showed that the U87 and U251 cells transfected with miR-18a inhibitor proliferated at lower levels than its negative control group (Po0.05). There was no obvious difference between untransfected group and miR-18a inhibitor NC group in the experiment. The U87 and U251 cells transfected for 48 h showed the highest inhibitory rate than the other three time point groups. Data were presented as mean7SD of three independent experiments. *Po0.05, vs. the miR-18a inhibitor NC group and #Po0.05, vs. the 24 h group.
Fig. 6 – MiR-18a inhibitor reduced migration and invasion in glioblastoma cells. U87 and U251 cells were transfected with miR-18a inhibitor and then subject to Transwell migration and invasion assays. After 24 h, migration and invasion cells were correspondingly counted after staining with 20% Giemsa. Representative photographs of migration and invasion cells on the membrane and accompanying statistical graphs were presented. The migration and invasion in U87 and U251 cells transfected with miR-18a inhibitor were significantly decreased compared with its negative control group (Po0.05). However, there was no obvious difference between untransfected group and miR-18a inhibitor NC group (P40.05). Data were presented as mean7SD of three independent experiments.*Po0.05, vs. the miR-18a inhibitor NC group. Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825
8
826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881 882 883 884 885
E XP E RI ME N TAL CE L L R ES E ARC H
] (]]]]) ]]]–]]]
Fig. 7 – MiR-18a inhibitor induced G0/G1 arrest in glioblastoma cells. The representative photographs showed cell cycle distribution of U87 and U251 cells in untransfected group, miR-18a inhibitor NC group and miR-18a inhibitor group. The percentage of cells in the different cell cycle phases was plotted, and we found that the percentage of cells transfected with miR-18a inhibitor distributed in G0/G1 phase was higher than its negative control group (Po0.05). And there was no obvious difference between untransfected group and miR-18a inhibitor NC group. Data were presented as mean7SD of three independent experiments. *Po0.05, vs. the miR-18a inhibitor NC group.
MiR-18a inhibitor promoted the apoptosis of glioblastoma cells The U87 and U251 cells were stained with Annexin V-FITC/PI and the apoptosis was determined by flow cytometry at 48 h after transfection (Fig. 8). The results demonstrated that the apoptosis rate in both cell lines transfected with miR-18a inhibitor was significantly increased compared with miR-18a inhibitor NC group (Po0.05), while there was no significant difference between untransfected group and miR-18a inhibitor NC group. In the rescue experiment, the apoptosis rate in both cell lines cotransfected with miR-18a and neogenin 30 -UTR was significantly decreased compared with that in the miR-18aþneogenin group (Po0.05; Fig. S3D).
Discussion In this study, we have found that the miR-18a expression was significantly increased in glioblastoma tissues and cell lines, while neogenin expression was clearly down-regulated. Furthermore, the expression levels of miR-18a and neogenin were directly and inversely (respectively) correlated to the pathological grade of glioma. MiR-18a negatively regulated the mRNA and protein expression of neogenin by binding to a specific site within the 30 -UTR of neogenin. Inhibition of miR-18a could remarkably alter the phenotype of human glioblastoma cells by inhibiting cellular proliferation, migration and invasion, inducing cell cycle arrest in G0/G1 phase, as well as promoting cellular apoptosis by upregulating the expression of neogenin. This is the first report, to date, outlining the phenotypic effects of miR-18a down-regulation in glioblastoma.
In recent years, increasing attention has been paid on the role of miRNAs in tumor development. MiR-18a, as an important member of miR-17-92 family, has shown various effects on different tumors. Firstly, as an oncogenic factor, miR-18a can inhibit the repair of damaged DNA by down-regulating the expression of ataxia telangiectasia mutated (ATM) in breast cancer cells [28]. MiR-18a in plasma also served as a novel potential marker in clinical screening or diagnosis for hepatitis B virus-related hepatocellular carcinoma, pancreatic cancer and esophageal squamous cell carcinoma [29–31]. Conversely, it was reported that miR-18a could act as a tumor suppressor as to inhibit the proliferation of T24 bladder cancer cells by down-regulating the expression of Dicer directly [32]. Therefore, the present research suggested that miR-18a could regulate various biological functions in different tumors by controlling target genes. In order to clarify the effects of miR-18a on glioblastoma, we first detected the expression of miR18a in collected clinical specimens and U87 and U251 human glioblastoma cell lines. The results showed that miR-18a expression was significantly up-regulated in glioblastoma tissues and cell lines compared with the human brain tissues and NHA. Furthermore, the expression level was increased with the rising pathological grades of glioblastoma, indicating that miR-18a might be an oncogene in glioblastoma. Our findings were consistent with previous studies, for example, Ernst reported that miR-18a expression in glioblastoma was up-regulated compared with normal brain tissues [33]. However, the underlying molecular mechanism relating the effects of miR-18a on glioblastoma still remains unclear. We attempted to predict the potential target genes of miR-18a in glioblastoma through applying the bioinformatics analysis method (TargetScan and Miranda), then we found neogenin, a theoretical target gene of miR-18a with the specific binding site in the 30 -UTR sequence.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944 945
E X PE R IM EN TA L C ELL R E S EA RC H
946 947 948 949 950 951 952 953 954 955 956 957 958 959 960 961 962 963 964 965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986 987 988 989 990 991 992 993 994 995 996 997 998 999 1000 1001 1002 1003 1004 1005
] (]]]]) ]]]–]]]
9
Fig. 8 – MiR-18a inhibitor promoted apoptosis in glioblastoma cells. Apoptosis of U87 and U251 cells was monitored by flow cytometry. Early apoptotic cells are Annexin Vþ/PI , late apoptotic cells are Annexin Vþ/PIþ, necrotic cells are Annexin V /PIþ and healthy cells are Annexin V /PI . The representative experiment of three groups was shown, and we found that the percentage of apoptotic cells in miR-18a inhibitor group was significantly increased compared with its negative control group (Po0.05). And there was no obvious difference between untransfected group and miR-18a inhibitor NC group. Data were presented as mean7SD of three independent experiments. *Po0.05, vs. the miR-18a inhibitor NC group.
Neogenin, highly similar to DCC in structure and function, plays an important role in the development of the central nervous system and has significant regulatory effects on a wide variety of biological activities such as formation of vessels and tissues as well as maintaining homeostasis after birth [34–36]. Recently neogenin was considered as a tumor suppressor in certain malignant tumors, such as lung adenocarcinomas [24], breast cancer [26], colon cancer [37] and prostate cancer [38] as the down-regulation of neogenin in these tumors could accelerate the tumor progression. Neogenin also acted as a tumor suppressor in glioblastoma because the down-regulation of its expression not only accelerated the tumorigenesis, but also influenced the migration and apoptosis of glioblastoma cells [27]. Nevertheless, the molecular mechanism underlying this process is still poorly understood. To clarify whether miR-18a could regulate the expression of neogenin and thereby influence the biological behavior of glioblastoma, we detected the neogenin expression level in glioblastoma. Results showed that neogenin expression was significantly down-regulated in glioblastoma tissues compared with the homologous surrounding non-neoplastic tissues, and the expression level was decreased with the rising pathological grades of glioblastoma. Wu et al. [27] reported that the distribution and expression of neogenin were narrower and lower in gliomas than their homologous surrounding areas, which was in accordance with our present work. Subsequently, we found that neogenin expression could be significantly up-regulated in the U87 and U251 cells by transfecting exogenous miR-18a inhibitor. Luciferase reporter assay also confirmed that miR-18a could target on the 30 -UTR of neogenin. Therefore, our results revealed the negative regulatory effects of miR-18a on neogenin expression in glioblastoma for the first time.
In our study, we also observed the inhibition of cellular proliferation, migration and invasion, induction of cell cycle arrest in the G0/G1 phase, as well as promotion of apoptosis in U87 and U251 cells by transfecting exogenous miR-18a inhibitor. In order to further understand the potential mechanism during the process, we co-transfected U87 and U251 cells with miR-18a expression vector and neogenin vectors with or without 30 -UTR. Results demonstrated that the effects of miR-18a on biological behavior of glioblastoma cells were related to the negative regulation of neogenin. We found that the expression level of neogenin was significantly down-regulated and cellular proliferation, migration and invasion were significantly recovered by co-transfection of miR-18a and neogenin with 30 -UTR. These data further validated the hypothesis that the changes of biological behavior in glioblastoma cells were due to the modulatory effects of miR-18a on neogenin. Therefore, inhibition of miR-18a could significantly suppress cell proliferation, migration and invasion and promote cellular apoptosis in glioblastoma cells by upregulating the expression of neogenin. Similarly, inhibition of miR-18a expression was reported to suppress these biological processes in other tumors as well, for example, inhibition of miR18a in neuroblastoma cells could lead to severe growth retardation by influencing the cell cycle [39]. Furthermore, recent studies also confirmed that the inhibition of other miRNAs could suppress the biological behavior of glioblastoma cells. Guessous et al. [40] found that inhibition of miR-10b expression significantly suppressed the cell proliferation, migration and invasion in glioblastoma cells and stem cells. Zhang et al. [41] found that inhibition of miR-106b expression could result in cell cycle arrest and suppress cell proliferation by up-regulating the expressions of p21 and RBL2 in U251 and LN229 glioblastoma cells. Taken these results
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
1006 1007 1008 1009 1010 1011 1012 1013 1014 1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 1046 1047 1048 1049 1050 1051 1052 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065
10
1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078 1079 1080 1081 1082 1083 1084 1085 1086 1087 1088 1089 1090 1091 1092 1093 1094 1095 1096 1097 1098 1099 1100 1101 1102 1103 1104 1105 1106 1107 1108 1109 1110 1111 1112 1113 1114 1115 1116 1117 1118 1119 1120 1121 1122 1123 1124 1125
E XP E RI ME N TAL CE L L R ES E ARC H
together, it is reasonable to infer that inhibiting the corresponding carcinogenic miRNAs might bring another therapeutic option for the treatment of malignant brain tumors. However, it is notable that even the same miRNA may produce the contrary effects in different tumors. For example, inhibition of miR-155 expression in glioblastoma U87 cells could significantly suppress the migration and invasion abilities and promote cellular apoptosis [42], whereas it could promote cell proliferation in human melanoma cells by up-regulating the expression of human SKI [43]. Therefore, how to inhibit the expression of correct target miRNAs precisely and specifically requires further investigation. In conclusion, this study revealed, for the first time, that inhibition of miR-18a expression could significantly suppress cell proliferation, migration and invasion, induce cell cycle arrest in G0/G1 phase and promote the apoptosis in glioblastoma U87 and U251 cells. The potential mechanism might be related to the increased levels of miR-18a-targeted neogenin. Therefore, as a potential novel target of glioblastoma, inhibition of miR-18a may provide a new therapeutic strategy for human glioblastoma treatment.
Acknowledgments Q2 This work is supported by grants from the Natural Science Founda-
tion of China (81172197, 81072056, 81171131, 81272564, and 81272795), the special fund for the Scientific Research of Doctordegree Subjects in Colleges and Universities (20102104110009), the Liaoning Science and Technology Plan Projects (No. 2011225020), the Shenyang Science and Technology Plan Projects (Nos. F12-277-1Q3 05, F13-316-1-16, and F13-220-9-15), and the Outstanding Scientific Fund of Shengjing Hospital.
Appendix A.
Supporting information
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.yexcr.2014.03. 009.
references [1] H. Ohgaki, Epidemiology of brain tumors, Methods Mol. Biol. 472 (2009) 323–342. [2] G. Li, Z.G. Zhang, Y.Y. Tu, T.B. Jin, H.J. Liang, Correlation of microrna-372 upregulation with poor prognosis in human glioma, Diagn. Pathol. 8 (2013) 1. [3] A. Stark, J. Brennecke, N. Bushati, R.B. Russell, S.M. Cohen, Animal microRNAs confer robustness to gene expression and have a significant impact on 30 UTR evolution, Cell 123 (2005) 1133–1146. [4] Y. Tian, Y. Nan, L. Han, A. Zhang, G. Wang, Z. Jia, J. Hao, P. Pu, Y. Zhong, C. Kang, MicroRNA miR-451 downregulates the PI3K/AKT pathway through CAB39 in human glioma, Int. J. Oncol. 40 (2012) 1105–1112. [5] S. Qu, Y. Yao, C. Shang, Y. Xue, J. Ma, Z. Li, Y. Liu, MicroRNA-330 is an oncogenic factor in glioblastoma cells by regulating SH3GL2 gene, PLoS One 7 (2012) e46010. [6] J.A. Chan, A.M. Krichevsky, K.S. Kosik, MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells, Cancer Res. 65 (2005) 6029–6033.
] (]]]]) ]]]–]]]
[7] Z. Lu, M. Liu, V. Stribinskis, C.M. Klinge, K.S. Ramos, N.H. Colburn, Y. Li, MicroRNA-21 promotes cell transformation by targeting the programmed cell death 4 gene, Oncogene 27 (2008) 4373–4379. [8] J.K. Gillies, I.A. Lorimer, Regulation of p27Kip1 by miRNA 221/222 in glioblastoma, Cell Cycle 6 (2007) 2005–2009. [9] Z.M. Shi, J. Wang, Z. Yan, Y.P. You, C.Y. Li, X. Qian, Y. Yin, P. Zhao, Y.Y. Wang, X.F. Wang, M.N. Li, L.Z. Liu, N. Liu, B.H. Jiang, MiR-128 inhibits tumor growth and angiogenesis by targeting p70S6K1, PLoS One 7 (2012) e32709. [10] J. Silber, A. Jacobsen, T. Ozawa, G. Harinath, A. Pedraza, C. Sander, E.C. Holland, J.T. Huse, MiR-34a repression in proneural malignant gliomas upregulates expression of its target PDGFRA and promotes tumorigenesis, PLoS One 7 (2012) e33844. [11] S. Takakura, N. Mitsutake, M. Nakashima, H. Namba, V.A. Saenko, T.I. Rogounovitch, Y. Nakazawa, T. Hayashi, A. Ohtsuru, S. Yamashita, Oncogenic role of miR-17-92 cluster in anaplastic thyroid cancer cells, Cancer Sci. 99 (2008) 1147–1154. [12] B. Diosdado, M.A. van de Wiel, J.S. Terhaar Sive Droste, S. Mongera, C. Postma, W.J. Meijerink, B. Carvalho, G.A. Meijer, MiR17-92 cluster is associated with 13q gain and c-myc expression during colorectal adenoma to adenocarcinoma progression, Br. J. Cancer 101 (2009) 707–714. [13] M. Karsy, E. Arslan, F. Moy, Current progress on understanding microRNAs in glioblastoma multiform, Genes Cancer 3 (2012) 3–15. [14] W.H. Liu, S.H. Yeh, C.C. Lu, S.L. Yu, H.Y. Chen, C.Y. Lin, D.S. Chen, P.J. Chen, MicroRNA-18a prevents estrogen receptor-alpha expression, promoting proliferation of hepatocellular carcinoma cells, Gastroenterology 136 (2009) 683–693. [15] W. Wu, M. Takanashi, N. Borjigin, S.I. Ohno, K. Fujita, S. Hoshino, Y. Osaka, A. Tsuchida, M. Kuroda, MicroRNA-18a modulates STAT3 activity through negative regulation of PIAS3 during gastricadenocarcinogenesis, Br. J. Cancer 108 (2013) 653–661. [16] L. Turchi, D.N. Debruyne, F. Almairac, V. Virolle, M. Fareh, Y. Neirijnck, F. Burel-Vandenbos, P. Paquis, M.P. Junier, E. Van Obberghen-Schilling, H. Chneiweiss, T. Virolle, Tumorigenic potential of miR-18An in glioma initiating cells requires NOTCH-1 signaling, Stem Cells 31 (2013) 1252–1265. [17] J.L. Fox, M. Dews, A.J. Minn, A. Thomas-Tikhonenko, Targeting of TGFβsignature andits essential component CTGF by miR-18 correlates with improved survival in glioblastoma, RNA 19 (2013) 177–190. [18] N.H. Wilson, B. Key, Neogenin: one receptor, many functions, Int. J. Biochem. Cell Biol. 39 (2007) 874–878. [19] S.J. Cole, D. Bradford, H.M. Cooper, Neogenin: a multi-functional receptor regulating diverse developmental processes, Int. J. Biochem. Cell Biol. 39 (2007) 1569–1575. [20] M. De Vries, H.M. Cooper, Emerging roles for neogenin and its ligands in CNS development, J. Neurochem. 106 (2008) 1483–1492. [21] E.R. Fearon, K.R. Cho, J.M. Nigro, S.E. Kern, J.W. Simons, J.M. Ruppert, S.R. Hamilton, A.C. Preisinger, G. Thomas, K.W. Kinzler, B. Vogelstein, Identification of a chromosome 18q gene that is altered in colorectal cancers, Science 247 (1990) 49–56. [22] S. Rodrigues, O. De Wever, E. Bruyneel, R.J. Rooney, C. Gespach, Opposing roles of netrin-1 and the dependence receptor DCC in cancer cell invasion, tumor growth and metastasis, Oncogene 26 (2007) 5615–5625. [23] K. Thiebault, L. Mazelin, L. Pays, F. Llambi, M.O. Joly, J.Y. Scoazec, J.C. Saurin, G. Romeo, P. Mehlen, The netrin-1 receptors UNC5H are putative tumor suppressors controlling cell death commitment, Proc. Natl. Acad. Sci. USA 100 (2003) 4173–4178. [24] P. Mehlen, C. Thibert, Dependence receptors: between life and death, Cell. Mol. Life Sci. 61 (2004) 1854–1866. [25] D. Berrar, B. Sturgeon, I. Bradbury, C.S. Downes, W. Dubitzky, Survival trees for analyzing clinical outcome in lung adenocarcinomas based on gene expression profiles: identification of neogenin and diacylglycerol kinase alpha expression as critical factors, J. Comput. Biol. 12 (2005) 534–544.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
1126 1127 1128 1129 1130 1131 1132 1133 1134 1135 1136 1137 1138 1139 1140 1141 1142 1143 1144 1145 1146 1147 1148 1149 1150 1151 1152 1153 1154 1155 1156 1157 1158 1159 1160 1161 1162 1163 1164 1165 1166 1167 1168 1169 1170 1171 1172 1173 1174 1175 1176 1177 1178 1179 1180 1181 1182 1183 1184 1185
E X PE R IM EN TA L C ELL R E S EA RC H
1186 1187 1188 1189 1190 1191 1192 1193 1194 1195 1196 1197 1198 1199 1200 1201 1202 1203 1204 1205 1206 1207 1208 1209 1210 1211 1212 1213 1214 1215 1216 1217 1218 1219 1220
[26] J.E. Lee, H.J. Kim, J.Y. Bae, S.W. Kim, J.S. Park, H.J. Shin, W. Han, S.W. Kim, K.S. Kang, D.Y. Noh, Neogenin expression may be inversely correlated to the tumorigenicity of human breast cancer, BMC Cancer 5 (2005) 154. [27] X. Wu, Y. Li, X. Wan, T.M. Kayira, R. Cao, X. Ju, X. Zhu, G. Zhao, Down-regulation of neogenin accelerated glioma progression through promoter methylation and its overexpression in SHG-44 induced apoptosis, PLoS One 7 (2012) e38074. [28] L. Song, C. Lin, Z. Wu, H. Gong, Y. Zeng, J. Wu, M. Li, J. Li, MiR-18a impairs DNA damage response through downregulation of ataxia telangiectasia mutated(ATM) kinase, PLoS One 6 (2011) e25454. [29] L. Li, Z. Guo, J. Wang, Y. Mao, Q. Gao, Serum miR-18a: a potential marker for hepatitis B virus-related hepatocellular carcinoma screening, Dig. Dis. Sci. 57 (2012) 2910–2916. [30] R. Morimura, S. Komatsu, D. Ichikawa, H. Takeshita, M. Tsujiura, H. Nagata, H. Konishi, A. Shiozaki, H. Ikoma, K. Okamoto, T. Ochiai, H. Taniguchi, E. Otsuji, Novel diagnostic value of circulating miR-18a in plasma of patients with pancreatic cancer, Br. J. Cancer 105 (2011) 1733–1740. [31] S. Hirajima, S. Komatsu, D. Ichikawa, H. Takeshita, H. Konishi, A. Shiozaki, R. Morimura, M. Tsujiura, H. Nagata, T. Kawaguchi, T. Arita, T. Kubota, H. Fujiwara, K. Okamoto, E. Otsuji, Clinical impact of circulating miR-18a in plasma of patients with oesophageal squamous cell carcinoma, Br. J. Cancer 108 (2013) 1822– 1829. [32] J. Tao, D. Wu, P. Li, B. Xu, Q. Lu, W. Zhang, MicroRNA-18a, a member of the oncogenic miR-17-92 cluster, targets Dicer and suppresses cell proliferation inbladder cancer T24 cells, Mol. Med. Rep. 5 (2012) 167–172. [33] A. Ernst, B. Campos, J. Meier, F. Devens, F. Liesenberg, M. Wolter, G. Reifenberger, C. Herold-Mende, P. Lichter, B. Radlwimmer, Derepression of CTGF via the miR-17-92 cluster upon differentiation of human glioblastoma spheroid cultures, Oncogene 29 (2010) 3411–3422. [34] Y. Han, Y. Shao, Z. Lin, Y.L. Qu, H. Wang, Y. Zhou, W. Chen, Y. Chen, W.L. Chen, F.R. Hu, W. Li, Z. Liu, Netrin-1 simultaneously suppresses corneal inflammation and neovascularization, Investig. Ophthalmol. Vis. Sci. 53 (2012) 1285–1295.
] (]]]]) ]]]–]]]
11
[35] T. Ito, M. Kagoshima, Y. Sasaki, C. Li, N. Udaka, T. Kitsukawa, H. Fujisawa, M. Taniguchi, T. Yagi, H. Kitamura, Y. Goshima, Repulsive axon guidance molecule Sema3A inhibits branching morphogenesis of fetal mouse lung, Mech. Dev. 97 (2000) 35–45. [36] C. Jayakumar, R. Mohamed, P.V. Ranganathan, G. Ramesh, Intracellular kinases mediate increased translation and secretion of netrin-1 from renal tubular epithelial cells, PLoS One 6 (2011) e26776. [37] V.S. Li, S.T. Yuen, T.L. Chan, H.H. Yan, W.L. Law, B.H. Yeung, A.S. Chan, W.Y. Tsui, S. So, X. Chen, S.Y. Leung, Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology 137 (2009) 176–187. [38] A. Latil, L. Chene, B. Cochant-Priollet, P. Mangin, G. Fournier, P. Berthon, O. Cussenot, Quantification of expression of netrins, slits and their receptors in human prostate tumors, Int. J. Cancer 103 (2003) 306–315. [39] J. Lovén, N. Zinin, T. Wahlström, I. Müller, P. Brodin, E. Fredlund, U. Ribacke, A. Pivarcsi, S. Påhlman, M. Henriksson, MYCNregulated microRNAs repress estrogen receptor-alpha (ESR1) expression and neuronal differentiation inhuman neuroblastoma, Proc. Natl. Acad. Sci. USA 107 (2010) 1553–1558. [40] F. Guessous, M. Alvarado-Velez, L. Marcinkiewicz, Y. Zhang, J. Kim, S. Heister, B. Kefas, J. Godlewski, D. Schiff, B. Purow, R. Abounader, Oncogenic effects of miR-10b in glioblastoma stem cells, J. Neurooncol. 112 (2013) 153–163. [41] A. Zhang, J. Hao, K. Wang, Q. Huang, K. Yu, C. Kang, G. Wang, Z. Jia, L. Han, P. Pu, Down-regulation of miR-106b suppresses the growth of human glioma cells, J. Neurooncol. 112 (2013) 179–189. [42] N. Ling, J. Gu, Z. Lei, M. Li, J. Zhao, H.T. Zhang, X. Li, microRNA-155 regulates cell proliferation and invasion by targeting FOXO3a in glioma, Oncol. Rep. 30 (2013) 2111–2118. [43] L. Levati, E. Pagani, S. Romani, D. Castiglia, E. Piccinni, C. Covaciu, P. Caporaso, S. Bondanza, F.R. Antonetti, E. Bonmassar, F. Martelli, E. Alvino, S. D'Atri, MicroRNA-155 targets the SKI gene in human melanoma cell lines, Pigment Cell Melanoma Res. 24 (2011) 538–550.
Please cite this article as: Y. Song, et al., MiR-18a regulates the proliferation, migration and invasion of human glioblastoma cell by targeting neogenin, Exp Cell Res (2014), http://dx.doi.org/10.1016/j.yexcr.2014.03.009
1221 1222 1223 1224 1225 1226 1227 1228 1229 1230 1231 1232 1233 1234 1235 1236 1237 1238 1239 1240 1241 1242 1243 1244 1245 1246 1247 1248 1249 1250 1251 1252 1253 1254