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miR-152 regulated glioma cell proliferation and apoptosis via Runx2 mediated by DNMT1
Biomedicine & Pharmacotherapy 92 (2017) 690–695
Available online at
ScienceDirect www.sciencedirect.com
Original article
miR-152 regulated glioma cell proliferation and apoptosis via Runx2 mediated by DNMT1 Peng Zhang* , Hongwei Sun, Bo Yang, Wenzheng Luo, Zengjin Liu, Junkuan Wang, Yuchao Zuo Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou, No. 1, East Jianshe Road, Zhengzhou, Henan Province, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2017 Received in revised form 17 May 2017 Accepted 22 May 2017
Background: Aberrant DNA methylation is associated with tumor onset and progression. Study has verified that the DNA methylation of miR-152 was mediated in many tumors, but whether it involved in glioblastomas was still unclear. Methods: This study enrolled 20 patients with glioma to analyze the expression pattern of miR-152. Real-time PCR and western blot were used to detect the mRNA or protein expression level, respectively. The relationship between miR-152 and runx2 was detected by Luciferase reporter assay. The methylation level of miR-152 was determined by methylation-specific PCR. Cell proliferation and apoptosis were detected by MTT and Annexin-FITC/PI assay. Results: The expression of miR-152 was down-regulated while the expression of DNMT1 was up-regulated in both glioma tissue and cell lines. MiR-152 was hypermethylated and its expression was negatively correlated with DNMT in glioma cell lines. DNMT1 knockdown promoted the expression of miR-152, however, DNMT1 overexpression suppressed the expression of miR-152. MiR-152 overexpression promoted glioma cell apoptosis while miR-152 knockdown promoted cell proliferation. MiR-152 targets Runx2 to regulate its expression, Runx2 overexpression abolished the effects of miR-152 overexpression. Conclusion: MiR-152 regulated cell proliferation and apoptosis of glioma mediated by Runx2, while the mechanism of down regulated miR-152 in glioma tissues and cells was its hypermethylation. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Glioma miR-152 Methylation Runx2 Apoptosis
1. Introduction Glioblastomas are the highly invasive brain tumors with high incidence and poor prognosis. Although many improvement has been made on the treatment, the strategies have not meet the needs of patients, the failure of treatment occurred in many patients due to the highly aggressive cancer. Therefore, to find new therapeutic strategy is necessary for the clinical treatment of gliomas. MiRNA is single-stranded RNA with the length of 18–22 nt that functioned as the regulators to govern genes expression in posttranscription process. Previous studies have verified that miRNAs bind to the 30 UTR of mRNA that lead to a reduction of protein. Based on these specific targeting, miRNAs regulate cell proliferation, apoptosis, signaling pathways and metabolic processes. In tumors, miRNA served as oncogene or tumor suppressor to manage tumor
* Corresponding author. E-mail address: [email protected] (P. Zhang). http://dx.doi.org/10.1016/j.biopha.2017.05.096 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
cell proliferation, invasion and apoptosis. For example, miR-26a acted as tumor suppressor by targeting Lin28B and Zcchc11 in melanoma, prostate cancer and liver cancer [1]. MiR-128 suppresses prostate cancer by targeting BMI-1 [2]. MiR-23b and miR-27b were the prognostic marker of clear cell renal cell carcinoma [3]. In addition, various miRNAs were selectively expressed in glioma, and their roles have been confirmed. For example, miR-146b acted as a biomarker to inhibit glioma growth [4]. miR-34a served as tumor suppressor that inhibit cell proliferation by targeting AKT and Wnt signaling [5]. MiR-152 has been widely studied in many cancers, such as breast cancer [6], non-small cell lung cancer [7], hepatocellular carcinoma [8] as well as glioma [9–11], the study confirmed that miR-152 served as tumor suppressor in glioma, but the mechanism of downregulated miR-152 in glioma was still unclear. DNA methylation is a reversible process of a nucleotide and mostly occurred in the 50 -C in CpG dinucleotides. The occurrence of DNA methylation could be influenced by many factors, such as genetic and environment factors. Studies have confirmed that DNA methylation play an important role in regulating genes and
P. Zhang et al. / Biomedicine & Pharmacotherapy 92 (2017) 690–695
miRNAs. Aberrant DNA methylation of miRNA has been found in many cancers, for example, DNA methylation of miR-124a is potential of colitis-associated cancer [12]. miR-128 methylation is associated with colorectal cancer [13]. Some of these miRNAs acted as tumor suppressors and aberrant DNA methylation is associated with increased malignancy or metastasis. Thus, to comprehensive analysis of DNA methylation of miRNA genes is important in developing of pathogenic mechanism. DNA methyltransferase inhibitors (DNMT) such as 5-AzadC (5-azacytidine) are effective in inhibiting methylation, recently, the drug has been approved by FDA on reversing of DNA methylation. Aberrant DNA methylation of miR-152 has been studied in endometrial cancer [14] and breast cancer [15], but whether the down-regulated miR-152 in glioma was mediated by DNA methylation was still unclear. In the present study, neuroglioma tissues or cells were obtained to explore the pathogenic mechanism of glioma, we further investigate the possible role of miR-152 on tumor cell proliferation and apoptosis, the results would provide potential foundation for further glioma therapy.
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2.2. Cell lines Human normal astrocytes NHA and neuroglioma cell lines U251,U87,A172 were purchased from American Type Culture Collection (ATCC). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, at 37 C in an incubator with 5% CO2. 2.3. Real-time PCR Total RNA was extracted from cells or tissues using Trizol (Invitrogen) according to the manufacture’s instruction. The RNA was quantified by agarose gel electrophoresis. Then 1 mg of DNA was taken out to reverse-transcript into cDNA using TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA). real-time PCR was carried out on an ABI 7900HT Fast Realtime PCR system by SYBR@ GREEN pcr Master Mix (Applied biosystems). NAPDH acted as internal control. The primers used in this study were synthesized by GENEWIZ Technologies Co., Ltd. (Suzhou, China)
2. Materials and methods 2.4. Western blot 2.1. Tissue samples A total of 20 neuroglioma tissue specimens and adjacent normal tissues were obtained from the tissue bank of The First Affiliated Hospital of Zhengzhou University, all tissues were saved in liquid nitrogen. The experiment were permitted by The First Affiliated Hospital of Zhengzhou University and all patients assigned informed consent prior to participation.
To assess protein expression of Runx2, western blot assay were performed. Briefly, cells were lysed by RIPA lysis buffer, the cellular protein were collected and the concentration were determined using a bicinchoninic acid assay kit (BCA) (Beyotime). Immunoblotting assay was conducted on a 12% SDS-PAGE gel, the equal amount of protein extract were blotted onto polyvinylidene fluoride membranes. The membrane was incubated by an antiRunx2 antibodies as well as 5% milk solution of Tris-buffered saline
Fig 1. MiR-152 was down-regulated and hypermethylated in glioma tissue and cells. A: the expression level of miR-152 in glioma tissue. B: real-time PCR determined the expression level of miR-152 in glioma cell lines. C: Methylation-specific PCR reflected the hypermethylation of miR-152 in glioma cell lines. D: 5-AzadC treatment pormoted the mRNA expression of miR-152 in glioma cell lines. D: 5-AzadC treatment suppressed the methylation level of miR-152 in glioma cell lines. Compared with Non-cancer, NHA or control, **P < 0.01.
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buffer at 4 C for 24 h. Then the membranes were incubated with secondary antibodies for another 2 h. The bands were observed by ECL method, b-actin served as internal control.
The Runx2 expression plasmid were purchased from Invitrogen (USA) and co-transfected into cells using Lipofectamine2000 reagent (Invitrogen) according to the manufacture’s instruction. The empty plasmid served as control.
2.5. Methylation-specific PCR analysis 2.7. MTT assay Genome DNA was extracted using CTAB method. Then 25 ng of bisulfite treated DNA was used for real-time PCR using an ABI 7900HT Fast Real-time PCR according to the manufacture’s instruction. The PCR products were purified by PCR Purification Kit (QIAGEN, Valencia, CA). The purified DNA was used as template for real-time PCR with nested primers, methylation-specific primers (MSP) and bisulfite-specific primers (BSP). The methylation level was calculated by sequencing the cloned MSP products.
Cell proliferation was measured by MTT assay using MTT Cell Proliferation Kit (Roche). Briefly, 1*105 cells were planted in a 96-well plate for 24 h cells were then incubated with MTT with 5% CO2, at 37 C for in a humidified atmosphere for 4 h. The absorbance was subsequently measured in 10 replicates at OD = 570 nm. 2.8. Annexin-FITC/PI apoptosis assay
2.6. Cell transfection Cells were maintained in a 96-well plate for 24 h the miR-152 mimic, inhibitor, as well as their negative control were transfected into cells using Lipofectamine2000 reagent (Invitrogen). The mimic, inhibitor, and negative control were all synthesized by GENEWIZ Technologies Co., Ltd. (Suzhou, China).
Cell apoptosis was measured by Annexin-FITC/PI assay. Briefly, cells were maintained in a 96-well plate, after 24 h, the cells were clearly washed with PBS and missed with 185 mL of 1X binding buffer. Then the cells were incubated with 5 mL of Annexin-V FITC and 10 mL of propidium iodide (PI) and maintained for 15 min in a dark environment. FACS calibur flowcytometer and Cell Quest Pro
Fig. 2. DNMT1 suppressed the expression of miR-152. A: the expression of DNMT1 in glioma tissue. B: the expression of miR-152 negative correlated with DNMT1 with R2 = 0.6274. C: the expression of DNMT1 in glioma cell lines was analyzed by real-time PCR and western blot. D: effects of DNMT1 knockdown and overexpression on DNMT1 expression in glioma cell line U251. E: effects of DNMT1 knockdown and overexpression on the expression of miR-152 in glioma cell line U251. F: effects of DNMT1 knockdown and overexpression on DNMT1 expression in glioma cell line U87. G: effects of DNMT1 knockdown and overexpression on the expression of miR-152 in glioma cell line U87. Compared with Non-cancer, NHA or si-NC, **P < 0.01.
P. Zhang et al. / Biomedicine & Pharmacotherapy 92 (2017) 690–695
software (Thermo Fisher) were used to measure the cell apoptosis. Flowcytometric data were analyzed using flow cytometry by FlowJo 7.6 software and displayed in dot plot of Annexin V/FITC-PI. 2.9. Luciferase reporter assay The sequence of Runx2 was searched in GenBank. Firefly luciferase cDNA fuse of Runx2 30 UTR was amplified and cloned into the specific vector to construct luciferase reporter plasmid (pGL-Runx2 or pGL-mutRunx2). The miR-152 mimic/inhibitor and pGL-Runx2/pGL-mutRunx2 were co-transfected into cells using Lipofectamine2000 reagent (Invitrogen) according to the manufacture’s instruction. Relative luciferase activity were determined by Dual-Luciferase Reporter Assay (Promega). 2.10. Statistical analysis All data were presented as means SD, the data were analyzed using SPASS 18.0 software. Statistical difference were processed using one-way analysis of variance (ANOVA) combined with t-test. P < 0.05 considered as statistically significant difference. 3. Results 3.1. Down-regulated miR-152 and high DNA methylation in glioma cell To detect the expression pattern of miR-152, a total of 20 brain glioma patients and normal control person were enrolled. Realtime PCR revealed that the expression of miR-152 was significantly
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decreased in the tissue of brain glioma patients rather than in normal control person (Fig. 1A). To determine the relationship between the expression and methylation of miR-152, we performed real-time PCR and DNA methylation analysis by using of neuroglioma cell lines U251, U87,A172 and the stellate cell line NHA (control). As is presented in Fig. 1B, the expression level of miR-152 was significantly lower in neuroglioma cell lines rather than in stellate cell line. While the DNA methylation of miR-152 was dramatically increased in neuroglioma cells than in control (Fig. 1C). Neuroglioma cell lines U251 and U87 pretreated by 10 mM 5-AzadC prominently increased the expression level (Fig. 1D), but decreased the DNA methylation of miR-152 (Fig. 1E) 3.2. The negative-correlation between DNMT1 and miR-152 To investigate the relationship between DNMT1 and miR-152, we first detect the expression level of DNMT1 in neuroglioma tissues from neuroglioma patients. Real-time PCR showed that the DNMT1 was dramatically increased in cancer tissues (Fig. 2A) and neuroglioma cells (Fig. 2C), Spearman’s rank correlation analysis presented that the expression levels of DNMT1 and miR-152 in neuroglioma tissue were inversely correlated (Fig. 2B). To confirm the relationship above, neuroglioma cells were transfected with siDNMT1 or pcDNA-DNMT1. We observed that DNMT1 knockdown remarkably decreased the expression of DNMT1 (Fig. 2D and F), increased the expression of miR-152 (Fig. 2E and G) in U251 and U87, While overexpressed DNMT1 dramatically increased the expression of DNMT1 (Fig. 2D and F), but decreased the expression of miR-152 in U251 and U87 (Fig. 2E and G).
Fig. 3. MiR-152 promoted cell apoptosis and suppressed cell proliferation in glioma cells. overexpressed miR-152 promoted the miR-152 expression (A), decreased cell proliferation (B) and increased cell apoptosis (C) in glioma cell line U251 and U87. MiR-152 knockdown inhibited miR-152 expression (D), promoted cell proliferation (E), and decreased cell apoptosis (F) in glioma cell line U251 and U87. Compared with pre-NC or NC, **P < 0.01.
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3.3. MiR-152 promoted cell proliferation in neuroglioma cells To investigate biological function of miR-152 in neuroglioma, we established adenovirus vector of miR-152. As was presented in Fig. 3A, overexpressed miR-152 promoted its expression, suppressed cell proliferation (Fig. 3B), accelerated cell apoptosis (Fig. 3C). In addition, cells of U251 and U87 transfected with miR152 inhibitor significantly decreased the expression of miR-152 (D),promoted cell proliferation (E), and inhibited cell apoptosis (F). 3.4. miR-152 targets runx2 to regulate its expression To identify the regulation mechanism of miR-152 on Runx2, we predicted the targets using an online database. Results revealed that miR-152 bind Runx2 To determine whether Runx2 was the target genes of miR-152, Runx2 30 UTR was cloned of the firefly luciferase reporter gene for the dual luciferase assay. The results showed that overexpressed miR-152 decreased pGL-Runx2 but not pGL-mutRunx2 activity, while down-regulated miR-152 promoted the activity of pGL-Runx2 but not pGL-mutRunx2 (Fig. 4A). Additionally, overexpressed miR-152 significantly decreased the mRNA and protein expression of Runx2, while down-regulated miR-152 promoted the mRNA and protein expression of Runx2 (Fig. 4B). To investigate the interaction between miR-152 and Runx2, cell proliferation and apoptosis were detected. We found that overexpressed miR-152 significantly decreased cell
proliferation, but increased cell apoptosis, while it was abolished by overexpressed Runx2 (Fig. 4C and D). 4. Discussion DNA methylation is an important adjustment process involved in developmental patterning, chromatin modification, and imprinting that influenced local transcriptional activity. Aberrant DNA methylation has been recognized to have an important role in disease processes. Aberrant DNA methylation of miRNA was the main reason that induced abnormal expressed miRNA. From this study, DNA methylation of miR-152 leading to the inhibition of miR-152 expression in glioma, while disruption of the regulatory circuit by DNA methyltransferase inhibitor 5-AzadC seemed to abrogate the effects and contribute to cancer suppression. DNA methylation was carried out by DNA methyltransferase enzymes, so far, the reported DNA methyltransferase enzymes including DNMT1, DNMT2, DNMT3. However, DNMT1 governs the methylation of existing genomic CpG island. Dysfunction of DNMT1 has been reported to favour of various different types of malignancies such as ovarian cancer [16], prostate cancer [17] and lung cancer [18]. Our study revealed that the levels of miR-152 and DNMT1 are negative correlated. In addition, DNMT1 regulated the methylation of miR-152, while the methylation of miR-152 regulated the expression of miR-152, namely, the expression of DNMT1 determines the level of miR-152. The results indicated that DNA
Fig. 4. MiR-152 regulated cell proliferation and apoptosis by mediating Runx2 in glioma cells. A: miR-152 targets Runx2 to regulate its expression. B: overexpressed miR-152 inhibited the expression of Runx2, while miR-152 knockdown promoted the expression of Runx2 in glioma cell lines U251 and U87. C: cell proliferation was determined by MTT assay with the treatment of miR-152 mimic and miR-152 mimic +pcDNA-Runx2. D: cell apoptosis was determined by Annexin-FITC/PI assay with the treatment of miR152 mimic and miR-152 mimic +pcDNA-Runx2. Compared with Pgl-Runx2 + miRNA control, pre-NC or NC, **P < 0.01.
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methylation acted as a regulator in glioma, and further glioma therapy aims on DNA methylation might be a new innovation thought. Runt-related transcription Factor 2 (Runx2) belongs to RUNX family (Runx1, Runx2, Runx3), which linked with various of cancers. At present, Runx2 has been identified to express in many tissues to regulate specific gene expression. For example, Runx2 was identified to mediate breast cancer-mediated bone metastasis [19]. In hepatocellular carcinoma, Runx2 promoted epithelialmesenchymal transition and vasculogenic mimicry formation [20]; miR-217 was identified to regulate cell proliferation, migration and apoptosis in glioma by targeting Runx2 [21]; moreover, Runx2 regulated galectin-3 in human glioma cells [22]. In this study, Runx2 was overexpressed in human glioma cells, and the expression of it related to cell proliferation and apoptosis in neuroglioma cells cooperatively. To better understand the role of miR-152 in neuroglioma, the underlying molecular mechanism was explored. We first identified the relationship between miR-152 and Runx2, luciferase reporter assay showed that Runx2 was the potential target gene of miR-152, miR-152 overexpression significantly inhibited the expression of Runx2, while miR-152 knock down prominently increased the expression of Runx2. Additionally, we also observed that miR-152 overexpression cell proliferation and promoted cell apoptosis, while the effects were abolished by overexpressed Runx2. In summary, we found that miR-152 was low expressed and Runx2 was highly expressed in neuroglioma. While the expression of miR-152 was mediated by DNA methylation, and the DNA methylation level was regulated by DNMT1. Our results finally confirmed that miR-152 was the indicative markers of glioma, moreover, the expression of Runx2 was an important prognostic factor. Additionally, the DNA methylation might relate to the onset and progression of neuroglioma. Acknowledgement This study was supported by the Health and Family Planning Commission of Henan Province (No. 201503076). References [1] X. Fu, et al., MiR-26a enhances miRNA biogenesis by targeting Lin28B and Zcchc11 to suppress tumor growth and metastasis, Oncogene 33 (34) (2014) 4296–4306.
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[2] M. Jin, et al., MiRNA-128 suppresses prostate cancer by inhibiting BMI-1 to inhibit tumor-initiating cells, Cancer Res. 74 (15) (2014) 4183–4195. [3] T. Ishihara, et al., Expression of the tumor suppressive miRNA-23b/27b cluster is a good prognostic marker in clear cell renal cell carcinoma, J. Urol. 192 (6) (2014) 1822–1830. [4] M. Katakowski, et al., Exosomes from marrow stromal cells expressing miR146b inhibit glioma growth, Cancer Lett. 335 (1) (2013) 201–204. [5] S.S. Rathod, et al., Tumor suppressive miRNA-34a suppresses cell proliferation and tumor growth of glioma stem cells by targeting Akt and Wnt signaling pathways, FEBS Open Biol. 4 (2014) 485–495. [6] E.A. Bachman, et al., Randomized controlled trial of benzocaine versus placebo spray for pain relief at hysterosalpingogram, Reprod. Biomed. Online 28 (6) (2014) 748–752. [7] J.S. Yang, et al., Serum miR-152: miR-148a, miR-148b, and miR-21 as novel biomarkers in non-small cell lung cancer screening, Tumour Biol. 36 (4) (2015) 3035–3042. [8] Y.W. Dang, et al., Effects of miR-152 on cell growth inhibition: motility suppression and apoptosis induction in hepatocellular carcinoma cells, Asian Pac. J. Cancer Prev. 15 (12) (2014) 4969–4976. [9] Y. Yao, et al., Knockdown of long non-coding RNA XIST exerts tumorsuppressive functions in human glioblastoma stem cells by up-regulating miR152, Cancer Lett. 359 (1) (2015) 75–86. [10] X. Zheng, et al., MiR-15b and miR-152 reduce glioma cell invasion and angiogenesis via NRP-2 and MMP-3, Cancer Lett. 329 (2) (2013) 146–154. [11] J. Ma, et al., MiR-152 functions as a tumor suppressor in glioblastoma stem cells by targeting Kruppel-like factor 4, Cancer Lett. 355 (1) (2014) 85–95. [12] Y. Ueda, et al., DNA methylation of microRNA-124a is a potential risk marker of colitis-associated cancer in patients with ulcerative colitis, Dig. Dis. Sci. 59 (10) (2014) 2444–2451. [13] Y. Takahashi, et al., Up-regulation of NEK2 by microRNA-128 methylation is associated with poor prognosis in colorectal cancer, Ann. Surg. Oncol. 21 (1) (2014) 205–212. [14] T. Tsuruta, et al., miR-152 is a tumor suppressor microRNA that is silenced by DNA hypermethylation in endometrial cancer, Cancer Res. 71 (20) (2011) 6450–6462. [15] D. Sengupta, et al., DNA methylation and not H3K4 trimethylation dictates the expression status of miR-152 gene which inhibits migration of breast cancer cells via DNMT1/CDH1 loop, Exp. Cell Res. 346 (2) (2016) 176–187. [16] Y. Xiang, et al., MiR-152 and miR-185 co-contribute to ovarian cancer cells cisplatin sensitivity by targeting DNMT1 directly: a novel epigenetic therapy independent of decitabine, Oncogene 33 (3) (2014) 378–386. [17] E. Lee, et al., DNMT1 regulates epithelial-mesenchymal transition and cancer stem cells: which promotes prostate cancer metastasis, Neoplasia 18 (9) (2016) 553–566. [18] S.A. Brodie, et al., Class I HDACs are mediators of smoke carcinogen-induced stabilization of DNMT1 and serve as promising targets for chemoprevention of lung cancer, Cancer Prev. Res. (Phila.) 7 (3) (2014) 351–361. [19] M. Vishal, et al., Role of Runx2 in breast cancer-mediated bone metastasis, Int. J. Biol. Macromol. 99 (2017) 608–614. [20] Z. Cao, et al., The expression and functional significance of runx2 in hepatocellular carcinoma: its role in vasculogenic mimicry and epithelialmesenchymal transition, Int. J. Mol. Sci. 18 (3) (2017). [21] Y. Zhu, et al., MicroRNA-217 inhibits cell proliferation and invasion by targeting Runx2 in human glioma, Am. J. Transl. Res. 8 (3) (2016) 1482–1491. [22] V. Vladimirova, et al., Runx2 is expressed in human glioma cells and mediates the expression of galectin-3, J. Neurosci. Res. 86 (11) (2008) 2450–2461.
Available online at
ScienceDirect www.sciencedirect.com
Original article
miR-152 regulated glioma cell proliferation and apoptosis via Runx2 mediated by DNMT1 Peng Zhang* , Hongwei Sun, Bo Yang, Wenzheng Luo, Zengjin Liu, Junkuan Wang, Yuchao Zuo Department of Neurosurgery, The First Affiliated Hospital of Zhengzhou, No. 1, East Jianshe Road, Zhengzhou, Henan Province, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 April 2017 Received in revised form 17 May 2017 Accepted 22 May 2017
Background: Aberrant DNA methylation is associated with tumor onset and progression. Study has verified that the DNA methylation of miR-152 was mediated in many tumors, but whether it involved in glioblastomas was still unclear. Methods: This study enrolled 20 patients with glioma to analyze the expression pattern of miR-152. Real-time PCR and western blot were used to detect the mRNA or protein expression level, respectively. The relationship between miR-152 and runx2 was detected by Luciferase reporter assay. The methylation level of miR-152 was determined by methylation-specific PCR. Cell proliferation and apoptosis were detected by MTT and Annexin-FITC/PI assay. Results: The expression of miR-152 was down-regulated while the expression of DNMT1 was up-regulated in both glioma tissue and cell lines. MiR-152 was hypermethylated and its expression was negatively correlated with DNMT in glioma cell lines. DNMT1 knockdown promoted the expression of miR-152, however, DNMT1 overexpression suppressed the expression of miR-152. MiR-152 overexpression promoted glioma cell apoptosis while miR-152 knockdown promoted cell proliferation. MiR-152 targets Runx2 to regulate its expression, Runx2 overexpression abolished the effects of miR-152 overexpression. Conclusion: MiR-152 regulated cell proliferation and apoptosis of glioma mediated by Runx2, while the mechanism of down regulated miR-152 in glioma tissues and cells was its hypermethylation. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Glioma miR-152 Methylation Runx2 Apoptosis
1. Introduction Glioblastomas are the highly invasive brain tumors with high incidence and poor prognosis. Although many improvement has been made on the treatment, the strategies have not meet the needs of patients, the failure of treatment occurred in many patients due to the highly aggressive cancer. Therefore, to find new therapeutic strategy is necessary for the clinical treatment of gliomas. MiRNA is single-stranded RNA with the length of 18–22 nt that functioned as the regulators to govern genes expression in posttranscription process. Previous studies have verified that miRNAs bind to the 30 UTR of mRNA that lead to a reduction of protein. Based on these specific targeting, miRNAs regulate cell proliferation, apoptosis, signaling pathways and metabolic processes. In tumors, miRNA served as oncogene or tumor suppressor to manage tumor
* Corresponding author. E-mail address: [email protected] (P. Zhang). http://dx.doi.org/10.1016/j.biopha.2017.05.096 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
cell proliferation, invasion and apoptosis. For example, miR-26a acted as tumor suppressor by targeting Lin28B and Zcchc11 in melanoma, prostate cancer and liver cancer [1]. MiR-128 suppresses prostate cancer by targeting BMI-1 [2]. MiR-23b and miR-27b were the prognostic marker of clear cell renal cell carcinoma [3]. In addition, various miRNAs were selectively expressed in glioma, and their roles have been confirmed. For example, miR-146b acted as a biomarker to inhibit glioma growth [4]. miR-34a served as tumor suppressor that inhibit cell proliferation by targeting AKT and Wnt signaling [5]. MiR-152 has been widely studied in many cancers, such as breast cancer [6], non-small cell lung cancer [7], hepatocellular carcinoma [8] as well as glioma [9–11], the study confirmed that miR-152 served as tumor suppressor in glioma, but the mechanism of downregulated miR-152 in glioma was still unclear. DNA methylation is a reversible process of a nucleotide and mostly occurred in the 50 -C in CpG dinucleotides. The occurrence of DNA methylation could be influenced by many factors, such as genetic and environment factors. Studies have confirmed that DNA methylation play an important role in regulating genes and
P. Zhang et al. / Biomedicine & Pharmacotherapy 92 (2017) 690–695
miRNAs. Aberrant DNA methylation of miRNA has been found in many cancers, for example, DNA methylation of miR-124a is potential of colitis-associated cancer [12]. miR-128 methylation is associated with colorectal cancer [13]. Some of these miRNAs acted as tumor suppressors and aberrant DNA methylation is associated with increased malignancy or metastasis. Thus, to comprehensive analysis of DNA methylation of miRNA genes is important in developing of pathogenic mechanism. DNA methyltransferase inhibitors (DNMT) such as 5-AzadC (5-azacytidine) are effective in inhibiting methylation, recently, the drug has been approved by FDA on reversing of DNA methylation. Aberrant DNA methylation of miR-152 has been studied in endometrial cancer [14] and breast cancer [15], but whether the down-regulated miR-152 in glioma was mediated by DNA methylation was still unclear. In the present study, neuroglioma tissues or cells were obtained to explore the pathogenic mechanism of glioma, we further investigate the possible role of miR-152 on tumor cell proliferation and apoptosis, the results would provide potential foundation for further glioma therapy.
691
2.2. Cell lines Human normal astrocytes NHA and neuroglioma cell lines U251,U87,A172 were purchased from American Type Culture Collection (ATCC). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, at 37 C in an incubator with 5% CO2. 2.3. Real-time PCR Total RNA was extracted from cells or tissues using Trizol (Invitrogen) according to the manufacture’s instruction. The RNA was quantified by agarose gel electrophoresis. Then 1 mg of DNA was taken out to reverse-transcript into cDNA using TaqMan miRNA reverse transcription kit (Applied Biosystems, Foster City, CA). real-time PCR was carried out on an ABI 7900HT Fast Realtime PCR system by SYBR@ GREEN pcr Master Mix (Applied biosystems). NAPDH acted as internal control. The primers used in this study were synthesized by GENEWIZ Technologies Co., Ltd. (Suzhou, China)
2. Materials and methods 2.4. Western blot 2.1. Tissue samples A total of 20 neuroglioma tissue specimens and adjacent normal tissues were obtained from the tissue bank of The First Affiliated Hospital of Zhengzhou University, all tissues were saved in liquid nitrogen. The experiment were permitted by The First Affiliated Hospital of Zhengzhou University and all patients assigned informed consent prior to participation.
To assess protein expression of Runx2, western blot assay were performed. Briefly, cells were lysed by RIPA lysis buffer, the cellular protein were collected and the concentration were determined using a bicinchoninic acid assay kit (BCA) (Beyotime). Immunoblotting assay was conducted on a 12% SDS-PAGE gel, the equal amount of protein extract were blotted onto polyvinylidene fluoride membranes. The membrane was incubated by an antiRunx2 antibodies as well as 5% milk solution of Tris-buffered saline
Fig 1. MiR-152 was down-regulated and hypermethylated in glioma tissue and cells. A: the expression level of miR-152 in glioma tissue. B: real-time PCR determined the expression level of miR-152 in glioma cell lines. C: Methylation-specific PCR reflected the hypermethylation of miR-152 in glioma cell lines. D: 5-AzadC treatment pormoted the mRNA expression of miR-152 in glioma cell lines. D: 5-AzadC treatment suppressed the methylation level of miR-152 in glioma cell lines. Compared with Non-cancer, NHA or control, **P < 0.01.
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P. Zhang et al. / Biomedicine & Pharmacotherapy 92 (2017) 690–695
buffer at 4 C for 24 h. Then the membranes were incubated with secondary antibodies for another 2 h. The bands were observed by ECL method, b-actin served as internal control.
The Runx2 expression plasmid were purchased from Invitrogen (USA) and co-transfected into cells using Lipofectamine2000 reagent (Invitrogen) according to the manufacture’s instruction. The empty plasmid served as control.
2.5. Methylation-specific PCR analysis 2.7. MTT assay Genome DNA was extracted using CTAB method. Then 25 ng of bisulfite treated DNA was used for real-time PCR using an ABI 7900HT Fast Real-time PCR according to the manufacture’s instruction. The PCR products were purified by PCR Purification Kit (QIAGEN, Valencia, CA). The purified DNA was used as template for real-time PCR with nested primers, methylation-specific primers (MSP) and bisulfite-specific primers (BSP). The methylation level was calculated by sequencing the cloned MSP products.
Cell proliferation was measured by MTT assay using MTT Cell Proliferation Kit (Roche). Briefly, 1*105 cells were planted in a 96-well plate for 24 h cells were then incubated with MTT with 5% CO2, at 37 C for in a humidified atmosphere for 4 h. The absorbance was subsequently measured in 10 replicates at OD = 570 nm. 2.8. Annexin-FITC/PI apoptosis assay
2.6. Cell transfection Cells were maintained in a 96-well plate for 24 h the miR-152 mimic, inhibitor, as well as their negative control were transfected into cells using Lipofectamine2000 reagent (Invitrogen). The mimic, inhibitor, and negative control were all synthesized by GENEWIZ Technologies Co., Ltd. (Suzhou, China).
Cell apoptosis was measured by Annexin-FITC/PI assay. Briefly, cells were maintained in a 96-well plate, after 24 h, the cells were clearly washed with PBS and missed with 185 mL of 1X binding buffer. Then the cells were incubated with 5 mL of Annexin-V FITC and 10 mL of propidium iodide (PI) and maintained for 15 min in a dark environment. FACS calibur flowcytometer and Cell Quest Pro
Fig. 2. DNMT1 suppressed the expression of miR-152. A: the expression of DNMT1 in glioma tissue. B: the expression of miR-152 negative correlated with DNMT1 with R2 = 0.6274. C: the expression of DNMT1 in glioma cell lines was analyzed by real-time PCR and western blot. D: effects of DNMT1 knockdown and overexpression on DNMT1 expression in glioma cell line U251. E: effects of DNMT1 knockdown and overexpression on the expression of miR-152 in glioma cell line U251. F: effects of DNMT1 knockdown and overexpression on DNMT1 expression in glioma cell line U87. G: effects of DNMT1 knockdown and overexpression on the expression of miR-152 in glioma cell line U87. Compared with Non-cancer, NHA or si-NC, **P < 0.01.
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software (Thermo Fisher) were used to measure the cell apoptosis. Flowcytometric data were analyzed using flow cytometry by FlowJo 7.6 software and displayed in dot plot of Annexin V/FITC-PI. 2.9. Luciferase reporter assay The sequence of Runx2 was searched in GenBank. Firefly luciferase cDNA fuse of Runx2 30 UTR was amplified and cloned into the specific vector to construct luciferase reporter plasmid (pGL-Runx2 or pGL-mutRunx2). The miR-152 mimic/inhibitor and pGL-Runx2/pGL-mutRunx2 were co-transfected into cells using Lipofectamine2000 reagent (Invitrogen) according to the manufacture’s instruction. Relative luciferase activity were determined by Dual-Luciferase Reporter Assay (Promega). 2.10. Statistical analysis All data were presented as means SD, the data were analyzed using SPASS 18.0 software. Statistical difference were processed using one-way analysis of variance (ANOVA) combined with t-test. P < 0.05 considered as statistically significant difference. 3. Results 3.1. Down-regulated miR-152 and high DNA methylation in glioma cell To detect the expression pattern of miR-152, a total of 20 brain glioma patients and normal control person were enrolled. Realtime PCR revealed that the expression of miR-152 was significantly
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decreased in the tissue of brain glioma patients rather than in normal control person (Fig. 1A). To determine the relationship between the expression and methylation of miR-152, we performed real-time PCR and DNA methylation analysis by using of neuroglioma cell lines U251, U87,A172 and the stellate cell line NHA (control). As is presented in Fig. 1B, the expression level of miR-152 was significantly lower in neuroglioma cell lines rather than in stellate cell line. While the DNA methylation of miR-152 was dramatically increased in neuroglioma cells than in control (Fig. 1C). Neuroglioma cell lines U251 and U87 pretreated by 10 mM 5-AzadC prominently increased the expression level (Fig. 1D), but decreased the DNA methylation of miR-152 (Fig. 1E) 3.2. The negative-correlation between DNMT1 and miR-152 To investigate the relationship between DNMT1 and miR-152, we first detect the expression level of DNMT1 in neuroglioma tissues from neuroglioma patients. Real-time PCR showed that the DNMT1 was dramatically increased in cancer tissues (Fig. 2A) and neuroglioma cells (Fig. 2C), Spearman’s rank correlation analysis presented that the expression levels of DNMT1 and miR-152 in neuroglioma tissue were inversely correlated (Fig. 2B). To confirm the relationship above, neuroglioma cells were transfected with siDNMT1 or pcDNA-DNMT1. We observed that DNMT1 knockdown remarkably decreased the expression of DNMT1 (Fig. 2D and F), increased the expression of miR-152 (Fig. 2E and G) in U251 and U87, While overexpressed DNMT1 dramatically increased the expression of DNMT1 (Fig. 2D and F), but decreased the expression of miR-152 in U251 and U87 (Fig. 2E and G).
Fig. 3. MiR-152 promoted cell apoptosis and suppressed cell proliferation in glioma cells. overexpressed miR-152 promoted the miR-152 expression (A), decreased cell proliferation (B) and increased cell apoptosis (C) in glioma cell line U251 and U87. MiR-152 knockdown inhibited miR-152 expression (D), promoted cell proliferation (E), and decreased cell apoptosis (F) in glioma cell line U251 and U87. Compared with pre-NC or NC, **P < 0.01.
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3.3. MiR-152 promoted cell proliferation in neuroglioma cells To investigate biological function of miR-152 in neuroglioma, we established adenovirus vector of miR-152. As was presented in Fig. 3A, overexpressed miR-152 promoted its expression, suppressed cell proliferation (Fig. 3B), accelerated cell apoptosis (Fig. 3C). In addition, cells of U251 and U87 transfected with miR152 inhibitor significantly decreased the expression of miR-152 (D),promoted cell proliferation (E), and inhibited cell apoptosis (F). 3.4. miR-152 targets runx2 to regulate its expression To identify the regulation mechanism of miR-152 on Runx2, we predicted the targets using an online database. Results revealed that miR-152 bind Runx2 To determine whether Runx2 was the target genes of miR-152, Runx2 30 UTR was cloned of the firefly luciferase reporter gene for the dual luciferase assay. The results showed that overexpressed miR-152 decreased pGL-Runx2 but not pGL-mutRunx2 activity, while down-regulated miR-152 promoted the activity of pGL-Runx2 but not pGL-mutRunx2 (Fig. 4A). Additionally, overexpressed miR-152 significantly decreased the mRNA and protein expression of Runx2, while down-regulated miR-152 promoted the mRNA and protein expression of Runx2 (Fig. 4B). To investigate the interaction between miR-152 and Runx2, cell proliferation and apoptosis were detected. We found that overexpressed miR-152 significantly decreased cell
proliferation, but increased cell apoptosis, while it was abolished by overexpressed Runx2 (Fig. 4C and D). 4. Discussion DNA methylation is an important adjustment process involved in developmental patterning, chromatin modification, and imprinting that influenced local transcriptional activity. Aberrant DNA methylation has been recognized to have an important role in disease processes. Aberrant DNA methylation of miRNA was the main reason that induced abnormal expressed miRNA. From this study, DNA methylation of miR-152 leading to the inhibition of miR-152 expression in glioma, while disruption of the regulatory circuit by DNA methyltransferase inhibitor 5-AzadC seemed to abrogate the effects and contribute to cancer suppression. DNA methylation was carried out by DNA methyltransferase enzymes, so far, the reported DNA methyltransferase enzymes including DNMT1, DNMT2, DNMT3. However, DNMT1 governs the methylation of existing genomic CpG island. Dysfunction of DNMT1 has been reported to favour of various different types of malignancies such as ovarian cancer [16], prostate cancer [17] and lung cancer [18]. Our study revealed that the levels of miR-152 and DNMT1 are negative correlated. In addition, DNMT1 regulated the methylation of miR-152, while the methylation of miR-152 regulated the expression of miR-152, namely, the expression of DNMT1 determines the level of miR-152. The results indicated that DNA
Fig. 4. MiR-152 regulated cell proliferation and apoptosis by mediating Runx2 in glioma cells. A: miR-152 targets Runx2 to regulate its expression. B: overexpressed miR-152 inhibited the expression of Runx2, while miR-152 knockdown promoted the expression of Runx2 in glioma cell lines U251 and U87. C: cell proliferation was determined by MTT assay with the treatment of miR-152 mimic and miR-152 mimic +pcDNA-Runx2. D: cell apoptosis was determined by Annexin-FITC/PI assay with the treatment of miR152 mimic and miR-152 mimic +pcDNA-Runx2. Compared with Pgl-Runx2 + miRNA control, pre-NC or NC, **P < 0.01.
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methylation acted as a regulator in glioma, and further glioma therapy aims on DNA methylation might be a new innovation thought. Runt-related transcription Factor 2 (Runx2) belongs to RUNX family (Runx1, Runx2, Runx3), which linked with various of cancers. At present, Runx2 has been identified to express in many tissues to regulate specific gene expression. For example, Runx2 was identified to mediate breast cancer-mediated bone metastasis [19]. In hepatocellular carcinoma, Runx2 promoted epithelialmesenchymal transition and vasculogenic mimicry formation [20]; miR-217 was identified to regulate cell proliferation, migration and apoptosis in glioma by targeting Runx2 [21]; moreover, Runx2 regulated galectin-3 in human glioma cells [22]. In this study, Runx2 was overexpressed in human glioma cells, and the expression of it related to cell proliferation and apoptosis in neuroglioma cells cooperatively. To better understand the role of miR-152 in neuroglioma, the underlying molecular mechanism was explored. We first identified the relationship between miR-152 and Runx2, luciferase reporter assay showed that Runx2 was the potential target gene of miR-152, miR-152 overexpression significantly inhibited the expression of Runx2, while miR-152 knock down prominently increased the expression of Runx2. Additionally, we also observed that miR-152 overexpression cell proliferation and promoted cell apoptosis, while the effects were abolished by overexpressed Runx2. In summary, we found that miR-152 was low expressed and Runx2 was highly expressed in neuroglioma. While the expression of miR-152 was mediated by DNA methylation, and the DNA methylation level was regulated by DNMT1. Our results finally confirmed that miR-152 was the indicative markers of glioma, moreover, the expression of Runx2 was an important prognostic factor. Additionally, the DNA methylation might relate to the onset and progression of neuroglioma. Acknowledgement This study was supported by the Health and Family Planning Commission of Henan Province (No. 201503076). References [1] X. Fu, et al., MiR-26a enhances miRNA biogenesis by targeting Lin28B and Zcchc11 to suppress tumor growth and metastasis, Oncogene 33 (34) (2014) 4296–4306.
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