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MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme
Biochemical and Biophysical Research Communications xxx (2015) 1e7
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MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme Shouping Dai a, Xianjun Wang b, Xiao Li c, Yuandong Cao d, * a
Department of Diagnostic Imaging, Linyi People's Hospital, Linyi, Shandong 276000, PR China Department of Neurology, Linyi People's Hospital, Linyi, Shandong 276000, PR China c Department of Pathology, First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China d Department of Radiotherapy, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 September 2015 Accepted 1 October 2015 Available online xxx
MicroRNA-139-5p was identified to be significantly down-regulated in glioblastoma multiform (GBM) by miRNA array. In this report we aimed to clarify its biological function, molecular mechanisms and direct target gene in GBM. Twelve patients with GBM were analyzed for the expression of miR-139-5p by quantitative RT-PCR. miR-139-5p overexpression was established by transfecting miR-139-5p-mimic into U87MG and T98G cells, and its effects on cell proliferation were studied using MTT assay and colony formation assays. We concluded that ectopic expression of miR-139-5p in GBM cell lines significantly suppressed cell proliferation and inducing apoptosis. Bioinformatics coupled with luciferase and western blot assays also revealed that miR-139-5p suppresses glioma cell proliferation by targeting ELTD1 and regulating cell cycle. © 2015 Elsevier Inc. All rights reserved.
Keywords: miR-139-5p Glioma ELTD1 Cell cycle
1. Introduction Glioma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans. Median survival without treatment is only approximately 4 months [1] and median survival with standard-of-care radiation and chemotherapy with temozolomide is only 15 months [2]. Despite the advent and development of treatment involving chemotherapy, radiation and surgery, the 5-year survival rate of GBM is less than 10%, with a final mortality rate of close to 100% [3,4]. Thus, new anticancer agents and therapeutic strategies are required to improve the patients live quality and prolong their life. Tumor cell migration and invasion of surrounding tissue are important characteristics of GBM. Cellular cytoskeleton remodeling and reduced cell and matrix adhesion also are factors often implicated in GBM invasion [5,6] and studies have shown various network proteins involved in the process [7e10]. Both ex vivo and in vivo validation studies indicate that ELTD1 (epidermal growth factor, latrophilin, and 7 transmembrane domain-containing protein 1 on chromosome 1) is a biomarker that can be used to confirm or detect the presence and grade of gliomas, particularly high-grade gliomas in humans and
this biomarker may play an important diagnostic role in addition to currently used markers for gliomas, particularly as a histological marker for identifying vascular proliferation [11]. Our results presented strongly suggested that the associative analysis method used in this study was able to accurately identify ELTD1 as a gliomaassociated biomarker, possibly due to increased angiogenesis. MicroRNAs (miRNAs) are a class of short (~22 nucleotides) noncoding RNA molecules that can regulate the expression of multiple targets by binding to complementary sequences located in the 30 untranslated regions (UTRs) of target mRNAs [12e14]. Recent advances have revealed that aberrant miRNA expression was implicated in pathogenesis of a variety of tumors and would be tested as potential biomarkers [15e17]. In order to find miRNAs that participate in the tumorigenesis of glioma, we performed a genome wide survey for microRNA expression and identified miR-139-5p reduced in human glioblastomas samples. miR-139 exerts function widely in human tumorigenesis and development. miR-139 was firstly reported in suppressing metastasis and progression of hepatocellular carcinoma by Wong's group [18]. Our study revealed that miR-139-5p acts as a tumor suppressor byinteracting with ELTD1 mRNA and regulating cell cycle in glioblastoma multiforme.
* Corresponding author. E-mail address: [email protected] (Y. Cao). http://dx.doi.org/10.1016/j.bbrc.2015.10.006 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: S. Dai, et al., MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.006
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2. Materials and methods 2.1. Patients All human tissue samples of normal brain and glioma were obtained from the Department of Neurosurgery, Linyi People's Hospital (Shandong, China). For the use of clinical specimens for research purposes, informed consent and approval were obtained from the Linyi People's Hospital Command of PLA. 2.2. Cell lines and cell culture Human glioblastoma cell lines U87MG, T98G were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, MD, USA) and antibiotics (50 U/ml penicillin, 50 mg/ml streptomycin) in a humidified atmosphere of CO2/air (5%/95%) at 37 C (Thermo Fisher Scientific, Nepean, Canada).
for 40 cycles. Data were analyzed by the Relative Quantification (DDCt) method using 7500 System SDS software (Applied Biosystems). The expression of each product was normalized to 18S rRNA and is shown as the ratio of the target gene to 18S gene expression, calculated by 2DDCt. 2.7. Flow cytometry analysis Cells were transfected with microRNAs for 6 h. After 48 h of incubation in DMEM complete medium, the cells were trypsinized and washed with cold NaCl/Pi, The cells were fixed in pre-chilled 70% ethanol at 20 C overnight. For measurement of DNA content, cells were stained with propidium iodide solution (50 ug/ml propidium iodide, 100 ug/mL RNase A, 0.05% Triton X-100 in NaCl/Pi), and incubated at 37 C in the dark for 30 min. DNA content was examined by flow cytometry using a FACSCalibur (BD Biosciences, San Jose, CA) with FLOWJO software (Tree Star, Ashland, OR). 2.8. Biotin-labeled RNA pull down assay
2.3. Western blot analysis and antibodies Whole-cell lysates were prepared as previously described [19]. The cell lysates were centrifuged and the protein concentration was evaluated with BCA protein assay (Pierce). Cellular proteins were extracted and separated in 4e10% Tris glycine/SDS-polyacrylamide gels and electrotransferred to ECL nitrocellulose membranes (#IPFL00010, Millipore). The membranes were blocked with 5% nonfat milk and incubated with specific antibodies. The b-actin protein was used as the endogenous control. Antibodies against the following proteins were purchased from Cell Signaling Technology: cyclin D1 (#2926), p21 (#2947), b-actin (#4967). The following antibodies were purchased from Santa Cruz Biotechnology: cyclin A (sc-239), cyclin E (sc-481), ELTD1 (sc-46947). Immunocomplexes were visualized by ECL (Pharmacia-Amersham, Freiburg, Germany). Bands intensity was determined by densitometry with BioRad Molecular Imager FX and Quantity One software. 2.4. Plasmids, transfection and gene reporter assay U87MG cells (8 104 cells/well) were seeded on 24-well plates 24 h before transfection. Cells were transfected using Lipofectamine™ 2000 Reagent (Invitrogen, CA, USA) with the plasmid carrying a firefly luciferase gene under promoter consisting multiple binding sites for miR139-5p. After cell lysis the luciferase activity was measured using Luciferase Reporter Assay System (Promega Corporation Madison, WI, USA) according to manufacturer's protocol.
U87MG and T98G cells were transfected with Bio-miR-139-5p or Bio-miR-control in two 60 mm dishes. After 48 h of incubation, the cells were trypsinized and washed twice with PBS. Cells were resuspended in 0.7 ml of lysis buffer (20 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.3% IGEPAL CA-630) and then incubated on ice for 20 min. The cytoplasmic lysate was isolated by centrifugation at 10,000 for 15 min and supernatant was collected. The lysate was added to the Strepatavidin-coated magnetic beads (Invitrogen) and incubated and incubated overnight at 4 C. The beads were washed with lysis buffer for 5 times and 100 ul of lysis buffer with DNaseI (2 U/ul) was added. After incubation at 37 C for 10 min, lysates were centrifuged at 5,000 g for 5 min and the supernatant was discarded. Protein kinase K (20 mg/ml) and 1ul of 10% SDS in 100 ul of lysis buffer were added to the pellet and incubated at 55 C for 20 min. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated with Trizol reagent (Invitrogen). The levels of ELTD1 in the Bio-miR-1395p pull-down were quantified by qRT-PCR. GAPDH was used for normalization. 2.9. Statistical analysis Each experiment was performed at least 3 times, on independent passages, usually in triplicates. Data were analyzed by NewmaneKeuls test using Statistica software as indicated and are presented as mean ± SEM. p < 0.05 was considered statistically significant. Results of time lapse microscopy experiments were analyzed with Wilcoxon test in R software.
2.5. Cell proliferation assay 3. Results U87MG cells (8 104 cells/well) were seeded in 24-well plates, incubated overnight and then MTT metabolism test was performed as previously described [20]. 2.6. Real-time PCR Total RNA (2 mg) isolated from U87MG cells transfected with plasmid coding for miR-139-5p was used as a template to generate cDNA. TaqMan MGB probe was marked with FAM™ reporter dye at the 50 end and nonfluorescent quencher at the 30 end of the probe. As endogenous control 18S (Hs99999901_s1) rRNA was applied. Gene expression quantification was performed using the Applied BiosystemTaqMan® Gene Expression Assay with the following parameters: stage 1 (50 C for 2 min) 1 cycle, stage 2 (95 C for 10 min) 1 cycle, stage 3 (95 C for 15 s, 60 C for 1 min)
3.1. miR-139-5p is downregulated in glioma cell lines and glioma tissues In a genome wide survey for microRNA expression, we have previously identified several microRNAs were reduced in human glioblastomas samples (unpublished results), including miR-1395p. To validate the result from our deep sequencing experiments, we examined miR-139-5p expression in various human tumor cell lines, total RNA was isolated from three human cell lines representing GBM (U87MG, LN-18 and A-172), pancreatic cancer (SW1990 and CFPAC-1), and prostate cancer (DU145 and PC-3) as well as from primary normal human astrocytes (NHA). Quantitative real time PCR (qPCR) was used to determine the mRNA level of miR139-5p. We found miR-139-5p was much lower in GBM cell lines as
Please cite this article in press as: S. Dai, et al., MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.006
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Fig. 1. Reduced levels of miR-139-5p expression in glioma cell lines and glioma tissues. (A) Real-time PCR analysis of miR-139-5p expression in primary normal human astrocytes (NHA) and various tumor cell lines. (B) Real-time PCR analysis of miR-139-5p expression in 5 nonneoplastic brain specimens and 12 human glioma tissues. The average miR-139-5p expression was normalized by U6 expression. Each bar represents the mean of three independent experiments. *P < 0.05. (C) Western blotting analysis of ELTD1 in primary normal human astrocytes (NHA) and glioma cell lines, b-actin served as the loading control.
compared with NHA cells (Fig. 1A). Furthermore, significant downregulation of miR-139-5p was also found in human glioblastomas samples compared with nonneoplastic brain specimens (Fig. 1B). Although it has been reported that ELTD1 ([epidermal growth factor (EGF), latrophilin and seven transmembrane
domain-containing 1] on chromosome 1) could be used as a putative glioma-associated marker (ELTD1, A Potential New Biomarker for Gliomas), we validated the ELTD1 protein level in NHA or cell lines representing GBM and found much higher reduction in NHA (Fig. 1C).
Fig. 2. miR-139-5p inhibits glioma cell proliferation and induces apoptosis. (A) Ectopic expression of miR-139-5p suppressed U87MG and T98G cell proliferation. The cell viability was determined by measuring MTT absorbance at A570. Cell growth was measured at every 24 h. NC represents negative control microRNA (means ± SD; **P < 0.01 compared to control, Student's t test). (B) Representative micrographs of crystal violet stained cell colonies. (C) Representative quantification of crystal violet stained cell colonies. (D) After transfection of miR-139-5p mimics or inhibitor to U87MG and T98G, the DNA content of PI-stained cells was analyzed by flow-cytometry. (E) Cells staining positive for Annexin VFITC and negative for PI at 48 h post-transfection were considered to have undergone apoptosis. (F) Average apoptotic rate of three independent experiments. Each bar represents the mean of three independent experiments. *P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: S. Dai, et al., MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.006
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Fig. 3. miR-139-5p regulates ELTD1 expression by binding 30 -UTR in GBM. (A) Western blot analysis. U87MG and T98G cells were transfected with miR-139-5p mimics after transfected with Dicer siRNA or negative control siRNA (N.C). The protein levels of ELTD1 and Dicer were detected with their specific antibodies. b-actin was used as the endogenous loading control. (B) Predicted miR-139-5p target sequence (blue) in the 30 UTR of ELTD1 (ELTD1-30 UTR) and positions of the mutated nucleotides (green) in the miR-139-5p (miR139-5p mut). Luciferase reporter assay of the indicated cell transfected with the pGL3-ELTD1-30 UTR reporter and 50 nM of miR-139-5p mimic and miR-139-5p mut oligonucleotides. (C) Luciferase reporter assay of the indicated cell transfected with pGL3-ELTD1-30 UTR or pGL3-ELTD1-30 UTR-mut reporter with 50 nM of miR-139 mimic. *P < 0.05. (D) The Biotinlabeled miR-139-5p-mRNA pull down assay. U87MG and T98G cells were transfected with Biotin-labeled microRNA control (Bio-NC) or Biotin-labeled miR-139-5p mimics for 48 h. The expressions of ELTD1 were measured by qRT-PCR and normalized to GAPDH (means ± SD; ***P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.2. miR-139-5p overexpression inhibits glioma cell proliferation and induces apoptosis To explore the role of miR-139-5p downregulation in the development and progression of glioma, we generated miR-1395p-mimic and miR-139-5p-inhibitor respectively. Transfected with miR-139-5p-mimic and miR-139-5p-inhibitor and negative control microRNA, we tried to reveal the gain-of-function effect or lose-of-function effect on the proliferation of the GBM cell lines. Results from the MTT (Methyl thiazolyltetrazolium) assay indicated that miR-139-5p upregulation significantly inhibited the proliferation rate of U87MG and T98G cells compared with the control while this situation would be rescued by the miR-139-5p suppression when cells were transfected by miR-139-5p-inhibitor (Fig. 2A). Results of crystal violet stained of cell colonies showed obvious reduction of cell proliferation in miR-139-5p overexpression cell lines (Fig. 2B). Same results were obtained from three independent repeats (Fig. 2C). In addition, the colony formation assays showed that U87MG and T98G glioma cells was significantly increased in response to miR-139 inhibitor (Fig. 2B, C). Furthermore, these results suggested that miR-139-5p upregulation inhibits glioma cell tumorigenicity in vitro. This result was
further confirmed by FACs analysis, which showed decreased the percentage of cells in S phase and increased the percentage of cells in G0/G1 phase in miR139-5p-overexpressing cells and at the same time we found that transfection of the miR-139-5p-inhibitor drastically increased the percentage of cells in the S peak but decreased the percentage of cells in the G0/G1 peak (Fig. 2D). Similar results obtained from U87MG and T98G cells. Double staining of the infected U87MG or T98G cells with annexin V-FITC and PI showed an obvious raise ratio of apoptosis in miR-139-5poverexpressing cells compared with the control cells while transfected with miR-139-5p-inhibitor can also rescue the apoptotic rate of cells both in U87MG and T98G cells (Fig. 2E). Same results were obtained from three independent repeats (Fig. 2F). Taken together, these results indicate that miR-139-5p suppresses glioma tumor cell proliferation and the proliferative effect of inhibiting miR-1395p in glioma cells may occur through regulation of G1/S transition. 3.3. miR-139-5p regulating ELTD1 by binding to the 30 -UTR in GBM To further evaluate how the miR-139-5p exerts tumor suppressor function in GBM, we used two publicly available algorithms bioinformatics algorithms (TargetScan and miRanda) and picked up
Please cite this article in press as: S. Dai, et al., MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.006
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Fig. 4. miR-139-5p inhibits cell proliferation through targeting ELTD1 30 UTR and regulating cell cycle related molecules in glioma. (A) Representative micrographs of crystal violet stained cell colonies. (B) Representative quantification of crystal violet stained cell colonies. *P < 0.05. (C) U87MG and T98G cells were transfected with miR-139-5p mimics or inhibitor. si-ELTD1 were used for knockdown of miR-139-5p target genes, respectively. The protein expression levels of G1/S regulatory molecules were analyzed by immunoblotting. NC represents negative control miRNA. (D) Co-transfection of miR-139-5p with 30 UTR-deleted ELTD1 plasmid (pME18s-ELTD1-FLAG) rescued the expressions of G1/S regulatory molecules. The expressions were analyzed by immunoblotting. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
ELTD1 mRNA as a theoretical target gene of miR-139-5p. Western blot assay showed that ELTD1 protein level was significantly downregulated in miR-139-5p-overexpressing cells (Fig. 3A). ELTD1 30 UTR contains the complementary binding sites of miR139-5p and it was cloned into a luciferase reporter vector to evaluate the influence of miR-139-5p on the expression of a reporter gene using a luciferase assay (Fig. 3B). We also generated the miR139-5p-mimic and miR-139-5p-inhibitor and miR-139-5p-mutant oligonucleotides and transfected them separately in U87MG or T98G cells to investigated the expression level of ELTD1 (Fig. 3B). We found that the expression of the reporter gene in the recombinant plasmid containing ELTD1 30 UTR was significantly attenuated in miR-139-5p-overexpressing cells and could be restored in miR-139-5p-mutant cells (Fig. 3B). Inhibition of miR-139-5p dramatically increased the expression of the reporter gene (Fig. 3B). To validate that miR-329 can directly bind to and regulate the levels of ELTD1 mRNA through the predicted binding sites, we altered bases of ELTD1 in the putative miR-139-5p binding site and found that the mutant 30 UTRs were completely refractory to miR139-5p-mediated luciferase reporter repression both in U87MG and T98G cells (Fig. 3C). Consistent with this observations, the Biotin-labeled miR-139-5p-mRNA pull down assay results also showed obvious reduction of the amount of ELTD1 protein in miR139-5p-overexpressing both in U87MG and T98G cells (Fig. 3D).
Collectively, our results demostrate that ELTD1 is a bona fide target of miR-139-5p and miR-139-5p regulate the expression of ELTD1 by directly targeting the 30 UTR of ELTD1. 3.4. miR-139-5p inhibits cell proliferation through regulating ELTD1 and cell cycle related molecules in glioma To investigate whether miR-139-5p inhibits cell proliferation was implicated with regulation of ELTD1, ELTD1 and ELTD1 30 UTR were respectively transfected into glioma cells with miR-139-5p overexpression using the Lipofectamine 2000 reagent. The result of colony formation assay showed overexpressing ELTD1 significantly increased the proliferation rate of U87MG and T98G glioma cells compared with that cells expressing ELTD1 30 UTR (Fig. 4A) and similar results were obtained from 3 independent repeats (Fig. 4B). The rescuing experiment further confirmed that the inhibitory role of miR-139-5p in glioma cells may be mediated by ELTD1. Our data showed that miR-139-5p exerts tumor suppressor function by regulating cell proliferation in GBM. So we examined some cell cycle regulator. Such as cyclin A, cyclin D1, cyclin E and p21, which has been implicated in the control of the G1 to S phase transition in mammals. Interestingly, we found the protein level of cyclin A and cyclin D1, a CDK regulator important for regulating the G1/S transition, was downregulated in U89MG and T98G glioma
Please cite this article in press as: S. Dai, et al., MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.006
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cells transfected with miR-139-5p-mimic, which is similar as transfected the si-ELDT1, but increased in the cells transfected with miR-139-5p-inhibitor, compared with control cells (Fig. 4C). On the other hand, the expression of p21 was increased in miR-139-5p overexpressing cells and inhibited in the miR-139-5p inhibited cells but the protein level of cyclin E was not affected obviously (Fig. 4C). Co-transfection of miR-139-5p with 30 UTR-deleted ELTD1 rescued the expressions of cyclin A and cyclin D1 while downregulating the p21 expression (Fig. 4D). 4. Discussion Glioma multiforme (GBM) is the most fatal form of all brain cancers in humans and so far there have been limited diagnostic tools. miRNAs play vital role in various biological processes, including proliferation, cellular differentiation, signal transduction and carcinogenesis. miRNAs regulate gene expression at the posttranscription level through inhibiting translation or promoting degradation of mRNAs by binding to complementary binding sites within the 30 untranslated region (30 UTR) of target messenger RNAs (mRNAs) [14]. So any disruption of microRNAs may cause disorder in gene regulating networks and cellular processes, particularly in oncogenesis and metastasis [21,22]. Therefore, the investigations of miRNAs and genes targeted by those miRNAs are likely to provide not only new insight into understanding the development of the GBM but also potential clinical therapeutic tools. miR-139-5p is one of the most pivotal miRNAs which were found to have extensive function in human tumorigenesis and development. It has been identified to have relevance with several tumor types and significantly downregulate, including gastric cancer, breast cancer, and GBM [23e25]. Consistent with previous study, our observations show that miR-139-5p degrades obviously in glioma cell lines U87MG, LN-18 and A-172. Furthermore, downregulation of miR-139-5p was also found in human glioblastomas samples. Previous studies have reported that miR-139 significantly inhibits oral cancer Tca8113 cells proliferation and induces cell apoptosis [26]. Further, our MTT assay and FACs analysis confirmed that forced expression of miR-139-5p inhibits glioma cell proliferation and promoting apoptosis. Our data shows that miR-139-5p interacts with ELTD1 by directly binding ELTD1 30 UTR. We firstly predicted ELTD1 as the substrate of miR-139-5p (Fig. 3A). Our Luciferase reporter assay and western blotting analysis consistently demonstrated that the protein level of ELTD1 is upregulated in glioma cells and negatively correlated with miR-139-5p expression (Figs. 1C and 4C). Our data showed the defect in G1/S phase transition in miR-1395p-overexpressing cells (Fig. 2C). Given the previous reports, we examined some cell cycle regulator molecules which are reported to be important for regulating the G1/S transition [27e31]. Our results showed that the important molecules in the regulation at G1/S transition, cyclin A and cyclin D1 were downregulated while p21 was upregulated in miR-139-5p-overexpressing cells. In summary, our analysis revealed that restoring miR-139-5p expression attenuated protein level of ELTD1 by binding the 30 UTR, and inhibited cell cycle progression in glioma. Targeting to the miR-139-5p/ELTD1 interaction or rescuing miR-139-5p expression may be a new therapeutic application to treat glioma patients in the future. Conflicts of interest The authors declare there is no conflicts of interest regarding the publication of this paper.
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MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme Shouping Dai a, Xianjun Wang b, Xiao Li c, Yuandong Cao d, * a
Department of Diagnostic Imaging, Linyi People's Hospital, Linyi, Shandong 276000, PR China Department of Neurology, Linyi People's Hospital, Linyi, Shandong 276000, PR China c Department of Pathology, First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China d Department of Radiotherapy, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 September 2015 Accepted 1 October 2015 Available online xxx
MicroRNA-139-5p was identified to be significantly down-regulated in glioblastoma multiform (GBM) by miRNA array. In this report we aimed to clarify its biological function, molecular mechanisms and direct target gene in GBM. Twelve patients with GBM were analyzed for the expression of miR-139-5p by quantitative RT-PCR. miR-139-5p overexpression was established by transfecting miR-139-5p-mimic into U87MG and T98G cells, and its effects on cell proliferation were studied using MTT assay and colony formation assays. We concluded that ectopic expression of miR-139-5p in GBM cell lines significantly suppressed cell proliferation and inducing apoptosis. Bioinformatics coupled with luciferase and western blot assays also revealed that miR-139-5p suppresses glioma cell proliferation by targeting ELTD1 and regulating cell cycle. © 2015 Elsevier Inc. All rights reserved.
Keywords: miR-139-5p Glioma ELTD1 Cell cycle
1. Introduction Glioma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor in humans. Median survival without treatment is only approximately 4 months [1] and median survival with standard-of-care radiation and chemotherapy with temozolomide is only 15 months [2]. Despite the advent and development of treatment involving chemotherapy, radiation and surgery, the 5-year survival rate of GBM is less than 10%, with a final mortality rate of close to 100% [3,4]. Thus, new anticancer agents and therapeutic strategies are required to improve the patients live quality and prolong their life. Tumor cell migration and invasion of surrounding tissue are important characteristics of GBM. Cellular cytoskeleton remodeling and reduced cell and matrix adhesion also are factors often implicated in GBM invasion [5,6] and studies have shown various network proteins involved in the process [7e10]. Both ex vivo and in vivo validation studies indicate that ELTD1 (epidermal growth factor, latrophilin, and 7 transmembrane domain-containing protein 1 on chromosome 1) is a biomarker that can be used to confirm or detect the presence and grade of gliomas, particularly high-grade gliomas in humans and
this biomarker may play an important diagnostic role in addition to currently used markers for gliomas, particularly as a histological marker for identifying vascular proliferation [11]. Our results presented strongly suggested that the associative analysis method used in this study was able to accurately identify ELTD1 as a gliomaassociated biomarker, possibly due to increased angiogenesis. MicroRNAs (miRNAs) are a class of short (~22 nucleotides) noncoding RNA molecules that can regulate the expression of multiple targets by binding to complementary sequences located in the 30 untranslated regions (UTRs) of target mRNAs [12e14]. Recent advances have revealed that aberrant miRNA expression was implicated in pathogenesis of a variety of tumors and would be tested as potential biomarkers [15e17]. In order to find miRNAs that participate in the tumorigenesis of glioma, we performed a genome wide survey for microRNA expression and identified miR-139-5p reduced in human glioblastomas samples. miR-139 exerts function widely in human tumorigenesis and development. miR-139 was firstly reported in suppressing metastasis and progression of hepatocellular carcinoma by Wong's group [18]. Our study revealed that miR-139-5p acts as a tumor suppressor byinteracting with ELTD1 mRNA and regulating cell cycle in glioblastoma multiforme.
* Corresponding author. E-mail address: [email protected] (Y. Cao). http://dx.doi.org/10.1016/j.bbrc.2015.10.006 0006-291X/© 2015 Elsevier Inc. All rights reserved.
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2. Materials and methods 2.1. Patients All human tissue samples of normal brain and glioma were obtained from the Department of Neurosurgery, Linyi People's Hospital (Shandong, China). For the use of clinical specimens for research purposes, informed consent and approval were obtained from the Linyi People's Hospital Command of PLA. 2.2. Cell lines and cell culture Human glioblastoma cell lines U87MG, T98G were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, MD, USA) and antibiotics (50 U/ml penicillin, 50 mg/ml streptomycin) in a humidified atmosphere of CO2/air (5%/95%) at 37 C (Thermo Fisher Scientific, Nepean, Canada).
for 40 cycles. Data were analyzed by the Relative Quantification (DDCt) method using 7500 System SDS software (Applied Biosystems). The expression of each product was normalized to 18S rRNA and is shown as the ratio of the target gene to 18S gene expression, calculated by 2DDCt. 2.7. Flow cytometry analysis Cells were transfected with microRNAs for 6 h. After 48 h of incubation in DMEM complete medium, the cells were trypsinized and washed with cold NaCl/Pi, The cells were fixed in pre-chilled 70% ethanol at 20 C overnight. For measurement of DNA content, cells were stained with propidium iodide solution (50 ug/ml propidium iodide, 100 ug/mL RNase A, 0.05% Triton X-100 in NaCl/Pi), and incubated at 37 C in the dark for 30 min. DNA content was examined by flow cytometry using a FACSCalibur (BD Biosciences, San Jose, CA) with FLOWJO software (Tree Star, Ashland, OR). 2.8. Biotin-labeled RNA pull down assay
2.3. Western blot analysis and antibodies Whole-cell lysates were prepared as previously described [19]. The cell lysates were centrifuged and the protein concentration was evaluated with BCA protein assay (Pierce). Cellular proteins were extracted and separated in 4e10% Tris glycine/SDS-polyacrylamide gels and electrotransferred to ECL nitrocellulose membranes (#IPFL00010, Millipore). The membranes were blocked with 5% nonfat milk and incubated with specific antibodies. The b-actin protein was used as the endogenous control. Antibodies against the following proteins were purchased from Cell Signaling Technology: cyclin D1 (#2926), p21 (#2947), b-actin (#4967). The following antibodies were purchased from Santa Cruz Biotechnology: cyclin A (sc-239), cyclin E (sc-481), ELTD1 (sc-46947). Immunocomplexes were visualized by ECL (Pharmacia-Amersham, Freiburg, Germany). Bands intensity was determined by densitometry with BioRad Molecular Imager FX and Quantity One software. 2.4. Plasmids, transfection and gene reporter assay U87MG cells (8 104 cells/well) were seeded on 24-well plates 24 h before transfection. Cells were transfected using Lipofectamine™ 2000 Reagent (Invitrogen, CA, USA) with the plasmid carrying a firefly luciferase gene under promoter consisting multiple binding sites for miR139-5p. After cell lysis the luciferase activity was measured using Luciferase Reporter Assay System (Promega Corporation Madison, WI, USA) according to manufacturer's protocol.
U87MG and T98G cells were transfected with Bio-miR-139-5p or Bio-miR-control in two 60 mm dishes. After 48 h of incubation, the cells were trypsinized and washed twice with PBS. Cells were resuspended in 0.7 ml of lysis buffer (20 mM Tris (pH 7.5), 100 mM KCl, 5 mM MgCl2, 0.3% IGEPAL CA-630) and then incubated on ice for 20 min. The cytoplasmic lysate was isolated by centrifugation at 10,000 for 15 min and supernatant was collected. The lysate was added to the Strepatavidin-coated magnetic beads (Invitrogen) and incubated and incubated overnight at 4 C. The beads were washed with lysis buffer for 5 times and 100 ul of lysis buffer with DNaseI (2 U/ul) was added. After incubation at 37 C for 10 min, lysates were centrifuged at 5,000 g for 5 min and the supernatant was discarded. Protein kinase K (20 mg/ml) and 1ul of 10% SDS in 100 ul of lysis buffer were added to the pellet and incubated at 55 C for 20 min. RNA bound to the beads (pull-down RNA) or from 10% of the extract (input RNA), was isolated with Trizol reagent (Invitrogen). The levels of ELTD1 in the Bio-miR-1395p pull-down were quantified by qRT-PCR. GAPDH was used for normalization. 2.9. Statistical analysis Each experiment was performed at least 3 times, on independent passages, usually in triplicates. Data were analyzed by NewmaneKeuls test using Statistica software as indicated and are presented as mean ± SEM. p < 0.05 was considered statistically significant. Results of time lapse microscopy experiments were analyzed with Wilcoxon test in R software.
2.5. Cell proliferation assay 3. Results U87MG cells (8 104 cells/well) were seeded in 24-well plates, incubated overnight and then MTT metabolism test was performed as previously described [20]. 2.6. Real-time PCR Total RNA (2 mg) isolated from U87MG cells transfected with plasmid coding for miR-139-5p was used as a template to generate cDNA. TaqMan MGB probe was marked with FAM™ reporter dye at the 50 end and nonfluorescent quencher at the 30 end of the probe. As endogenous control 18S (Hs99999901_s1) rRNA was applied. Gene expression quantification was performed using the Applied BiosystemTaqMan® Gene Expression Assay with the following parameters: stage 1 (50 C for 2 min) 1 cycle, stage 2 (95 C for 10 min) 1 cycle, stage 3 (95 C for 15 s, 60 C for 1 min)
3.1. miR-139-5p is downregulated in glioma cell lines and glioma tissues In a genome wide survey for microRNA expression, we have previously identified several microRNAs were reduced in human glioblastomas samples (unpublished results), including miR-1395p. To validate the result from our deep sequencing experiments, we examined miR-139-5p expression in various human tumor cell lines, total RNA was isolated from three human cell lines representing GBM (U87MG, LN-18 and A-172), pancreatic cancer (SW1990 and CFPAC-1), and prostate cancer (DU145 and PC-3) as well as from primary normal human astrocytes (NHA). Quantitative real time PCR (qPCR) was used to determine the mRNA level of miR139-5p. We found miR-139-5p was much lower in GBM cell lines as
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Fig. 1. Reduced levels of miR-139-5p expression in glioma cell lines and glioma tissues. (A) Real-time PCR analysis of miR-139-5p expression in primary normal human astrocytes (NHA) and various tumor cell lines. (B) Real-time PCR analysis of miR-139-5p expression in 5 nonneoplastic brain specimens and 12 human glioma tissues. The average miR-139-5p expression was normalized by U6 expression. Each bar represents the mean of three independent experiments. *P < 0.05. (C) Western blotting analysis of ELTD1 in primary normal human astrocytes (NHA) and glioma cell lines, b-actin served as the loading control.
compared with NHA cells (Fig. 1A). Furthermore, significant downregulation of miR-139-5p was also found in human glioblastomas samples compared with nonneoplastic brain specimens (Fig. 1B). Although it has been reported that ELTD1 ([epidermal growth factor (EGF), latrophilin and seven transmembrane
domain-containing 1] on chromosome 1) could be used as a putative glioma-associated marker (ELTD1, A Potential New Biomarker for Gliomas), we validated the ELTD1 protein level in NHA or cell lines representing GBM and found much higher reduction in NHA (Fig. 1C).
Fig. 2. miR-139-5p inhibits glioma cell proliferation and induces apoptosis. (A) Ectopic expression of miR-139-5p suppressed U87MG and T98G cell proliferation. The cell viability was determined by measuring MTT absorbance at A570. Cell growth was measured at every 24 h. NC represents negative control microRNA (means ± SD; **P < 0.01 compared to control, Student's t test). (B) Representative micrographs of crystal violet stained cell colonies. (C) Representative quantification of crystal violet stained cell colonies. (D) After transfection of miR-139-5p mimics or inhibitor to U87MG and T98G, the DNA content of PI-stained cells was analyzed by flow-cytometry. (E) Cells staining positive for Annexin VFITC and negative for PI at 48 h post-transfection were considered to have undergone apoptosis. (F) Average apoptotic rate of three independent experiments. Each bar represents the mean of three independent experiments. *P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. miR-139-5p regulates ELTD1 expression by binding 30 -UTR in GBM. (A) Western blot analysis. U87MG and T98G cells were transfected with miR-139-5p mimics after transfected with Dicer siRNA or negative control siRNA (N.C). The protein levels of ELTD1 and Dicer were detected with their specific antibodies. b-actin was used as the endogenous loading control. (B) Predicted miR-139-5p target sequence (blue) in the 30 UTR of ELTD1 (ELTD1-30 UTR) and positions of the mutated nucleotides (green) in the miR-139-5p (miR139-5p mut). Luciferase reporter assay of the indicated cell transfected with the pGL3-ELTD1-30 UTR reporter and 50 nM of miR-139-5p mimic and miR-139-5p mut oligonucleotides. (C) Luciferase reporter assay of the indicated cell transfected with pGL3-ELTD1-30 UTR or pGL3-ELTD1-30 UTR-mut reporter with 50 nM of miR-139 mimic. *P < 0.05. (D) The Biotinlabeled miR-139-5p-mRNA pull down assay. U87MG and T98G cells were transfected with Biotin-labeled microRNA control (Bio-NC) or Biotin-labeled miR-139-5p mimics for 48 h. The expressions of ELTD1 were measured by qRT-PCR and normalized to GAPDH (means ± SD; ***P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.2. miR-139-5p overexpression inhibits glioma cell proliferation and induces apoptosis To explore the role of miR-139-5p downregulation in the development and progression of glioma, we generated miR-1395p-mimic and miR-139-5p-inhibitor respectively. Transfected with miR-139-5p-mimic and miR-139-5p-inhibitor and negative control microRNA, we tried to reveal the gain-of-function effect or lose-of-function effect on the proliferation of the GBM cell lines. Results from the MTT (Methyl thiazolyltetrazolium) assay indicated that miR-139-5p upregulation significantly inhibited the proliferation rate of U87MG and T98G cells compared with the control while this situation would be rescued by the miR-139-5p suppression when cells were transfected by miR-139-5p-inhibitor (Fig. 2A). Results of crystal violet stained of cell colonies showed obvious reduction of cell proliferation in miR-139-5p overexpression cell lines (Fig. 2B). Same results were obtained from three independent repeats (Fig. 2C). In addition, the colony formation assays showed that U87MG and T98G glioma cells was significantly increased in response to miR-139 inhibitor (Fig. 2B, C). Furthermore, these results suggested that miR-139-5p upregulation inhibits glioma cell tumorigenicity in vitro. This result was
further confirmed by FACs analysis, which showed decreased the percentage of cells in S phase and increased the percentage of cells in G0/G1 phase in miR139-5p-overexpressing cells and at the same time we found that transfection of the miR-139-5p-inhibitor drastically increased the percentage of cells in the S peak but decreased the percentage of cells in the G0/G1 peak (Fig. 2D). Similar results obtained from U87MG and T98G cells. Double staining of the infected U87MG or T98G cells with annexin V-FITC and PI showed an obvious raise ratio of apoptosis in miR-139-5poverexpressing cells compared with the control cells while transfected with miR-139-5p-inhibitor can also rescue the apoptotic rate of cells both in U87MG and T98G cells (Fig. 2E). Same results were obtained from three independent repeats (Fig. 2F). Taken together, these results indicate that miR-139-5p suppresses glioma tumor cell proliferation and the proliferative effect of inhibiting miR-1395p in glioma cells may occur through regulation of G1/S transition. 3.3. miR-139-5p regulating ELTD1 by binding to the 30 -UTR in GBM To further evaluate how the miR-139-5p exerts tumor suppressor function in GBM, we used two publicly available algorithms bioinformatics algorithms (TargetScan and miRanda) and picked up
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Fig. 4. miR-139-5p inhibits cell proliferation through targeting ELTD1 30 UTR and regulating cell cycle related molecules in glioma. (A) Representative micrographs of crystal violet stained cell colonies. (B) Representative quantification of crystal violet stained cell colonies. *P < 0.05. (C) U87MG and T98G cells were transfected with miR-139-5p mimics or inhibitor. si-ELTD1 were used for knockdown of miR-139-5p target genes, respectively. The protein expression levels of G1/S regulatory molecules were analyzed by immunoblotting. NC represents negative control miRNA. (D) Co-transfection of miR-139-5p with 30 UTR-deleted ELTD1 plasmid (pME18s-ELTD1-FLAG) rescued the expressions of G1/S regulatory molecules. The expressions were analyzed by immunoblotting. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
ELTD1 mRNA as a theoretical target gene of miR-139-5p. Western blot assay showed that ELTD1 protein level was significantly downregulated in miR-139-5p-overexpressing cells (Fig. 3A). ELTD1 30 UTR contains the complementary binding sites of miR139-5p and it was cloned into a luciferase reporter vector to evaluate the influence of miR-139-5p on the expression of a reporter gene using a luciferase assay (Fig. 3B). We also generated the miR139-5p-mimic and miR-139-5p-inhibitor and miR-139-5p-mutant oligonucleotides and transfected them separately in U87MG or T98G cells to investigated the expression level of ELTD1 (Fig. 3B). We found that the expression of the reporter gene in the recombinant plasmid containing ELTD1 30 UTR was significantly attenuated in miR-139-5p-overexpressing cells and could be restored in miR-139-5p-mutant cells (Fig. 3B). Inhibition of miR-139-5p dramatically increased the expression of the reporter gene (Fig. 3B). To validate that miR-329 can directly bind to and regulate the levels of ELTD1 mRNA through the predicted binding sites, we altered bases of ELTD1 in the putative miR-139-5p binding site and found that the mutant 30 UTRs were completely refractory to miR139-5p-mediated luciferase reporter repression both in U87MG and T98G cells (Fig. 3C). Consistent with this observations, the Biotin-labeled miR-139-5p-mRNA pull down assay results also showed obvious reduction of the amount of ELTD1 protein in miR139-5p-overexpressing both in U87MG and T98G cells (Fig. 3D).
Collectively, our results demostrate that ELTD1 is a bona fide target of miR-139-5p and miR-139-5p regulate the expression of ELTD1 by directly targeting the 30 UTR of ELTD1. 3.4. miR-139-5p inhibits cell proliferation through regulating ELTD1 and cell cycle related molecules in glioma To investigate whether miR-139-5p inhibits cell proliferation was implicated with regulation of ELTD1, ELTD1 and ELTD1 30 UTR were respectively transfected into glioma cells with miR-139-5p overexpression using the Lipofectamine 2000 reagent. The result of colony formation assay showed overexpressing ELTD1 significantly increased the proliferation rate of U87MG and T98G glioma cells compared with that cells expressing ELTD1 30 UTR (Fig. 4A) and similar results were obtained from 3 independent repeats (Fig. 4B). The rescuing experiment further confirmed that the inhibitory role of miR-139-5p in glioma cells may be mediated by ELTD1. Our data showed that miR-139-5p exerts tumor suppressor function by regulating cell proliferation in GBM. So we examined some cell cycle regulator. Such as cyclin A, cyclin D1, cyclin E and p21, which has been implicated in the control of the G1 to S phase transition in mammals. Interestingly, we found the protein level of cyclin A and cyclin D1, a CDK regulator important for regulating the G1/S transition, was downregulated in U89MG and T98G glioma
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cells transfected with miR-139-5p-mimic, which is similar as transfected the si-ELDT1, but increased in the cells transfected with miR-139-5p-inhibitor, compared with control cells (Fig. 4C). On the other hand, the expression of p21 was increased in miR-139-5p overexpressing cells and inhibited in the miR-139-5p inhibited cells but the protein level of cyclin E was not affected obviously (Fig. 4C). Co-transfection of miR-139-5p with 30 UTR-deleted ELTD1 rescued the expressions of cyclin A and cyclin D1 while downregulating the p21 expression (Fig. 4D). 4. Discussion Glioma multiforme (GBM) is the most fatal form of all brain cancers in humans and so far there have been limited diagnostic tools. miRNAs play vital role in various biological processes, including proliferation, cellular differentiation, signal transduction and carcinogenesis. miRNAs regulate gene expression at the posttranscription level through inhibiting translation or promoting degradation of mRNAs by binding to complementary binding sites within the 30 untranslated region (30 UTR) of target messenger RNAs (mRNAs) [14]. So any disruption of microRNAs may cause disorder in gene regulating networks and cellular processes, particularly in oncogenesis and metastasis [21,22]. Therefore, the investigations of miRNAs and genes targeted by those miRNAs are likely to provide not only new insight into understanding the development of the GBM but also potential clinical therapeutic tools. miR-139-5p is one of the most pivotal miRNAs which were found to have extensive function in human tumorigenesis and development. It has been identified to have relevance with several tumor types and significantly downregulate, including gastric cancer, breast cancer, and GBM [23e25]. Consistent with previous study, our observations show that miR-139-5p degrades obviously in glioma cell lines U87MG, LN-18 and A-172. Furthermore, downregulation of miR-139-5p was also found in human glioblastomas samples. Previous studies have reported that miR-139 significantly inhibits oral cancer Tca8113 cells proliferation and induces cell apoptosis [26]. Further, our MTT assay and FACs analysis confirmed that forced expression of miR-139-5p inhibits glioma cell proliferation and promoting apoptosis. Our data shows that miR-139-5p interacts with ELTD1 by directly binding ELTD1 30 UTR. We firstly predicted ELTD1 as the substrate of miR-139-5p (Fig. 3A). Our Luciferase reporter assay and western blotting analysis consistently demonstrated that the protein level of ELTD1 is upregulated in glioma cells and negatively correlated with miR-139-5p expression (Figs. 1C and 4C). Our data showed the defect in G1/S phase transition in miR-1395p-overexpressing cells (Fig. 2C). Given the previous reports, we examined some cell cycle regulator molecules which are reported to be important for regulating the G1/S transition [27e31]. Our results showed that the important molecules in the regulation at G1/S transition, cyclin A and cyclin D1 were downregulated while p21 was upregulated in miR-139-5p-overexpressing cells. In summary, our analysis revealed that restoring miR-139-5p expression attenuated protein level of ELTD1 by binding the 30 UTR, and inhibited cell cycle progression in glioma. Targeting to the miR-139-5p/ELTD1 interaction or rescuing miR-139-5p expression may be a new therapeutic application to treat glioma patients in the future. Conflicts of interest The authors declare there is no conflicts of interest regarding the publication of this paper.
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Please cite this article in press as: S. Dai, et al., MicroRNA-139-5p acts as a tumor suppressor by targeting ELTD1 and regulating cell cycle in glioblastoma multiforme, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/10.1016/j.bbrc.2015.10.006