European Review for Medical and Pharmacological Sciences 2017; 21: 5630-5637 MiR-616 promotes proliferation and inhibits apoptosis in glioma cells by suppressing expression of SOX7 via the

Q.-L. BAI, C.-W. HU, X.-R. WANG, J.-X. SHANG, G.-F. YIN

Department of Neurosurgery, Cangzhou Central Hospital, Cangzhou, Hebei, China

Abstract. – OBJECTIVE: Accumulating ev- cells in the brain2. In 2007, the World Health Or- idence has indicated that miR-616 exerts tumor ganization (WHO) categorized gliomas into four promoter roles in several types of cancer. Howev- grades of severity, I to IV3. Glioblastoma multi- er, the expression pattern and roles of miR-616 in form (GBM, WHO grade IV) is the most com- glioma progression remain unknown. This study aimed to reveal the role of miR-616 in glioma cell mon subtype of glioma. The prognosis of patients proliferation and its potential mechanisms. with GBM is extremely poor despite multimodal PATIENTS AND METHODS: Real-time poly- treatments, including surgery, chemotherapy and merase chain reaction was used to assay the radiotherapy4. Therefore, there is a great need for expression of miR-616 in glioma tissue sam- understanding the underlying biological mecha- ples and glioma cell lines. MTT proliferation as- nism to found new biomarkers and targeted thera- say and flow cytometry analysis were performed pies for this disease. to test the apoptosis and proliferation of glioma cell after down-regulation of miR-616. The tar- MicroRNAs (miRNAs) are a class of endoge- get of miR-616 was predicted by TargetScan and nous small noncoding single-stranded RNA mole- confirmed by luciferase reporter assay. Chang- cules, about 22 nucleotides in length5. They regu- es in Wnt signaling markers expression were as- late expression by targeting -encoding sessed using Western blotting. mRNA. Growing evidence has shown that diverse RESULTS: We found that the expression of biological processes, such as cell proliferation, dif- miR-616 was increased in glioma tissues and cell ferentiation, apoptosis, fat metabolism and onco- lines. MTT and low cytometry analysis indicated 6,7 that down-regulation of miR-616 significantly in- genesis, were under the regulation of miRNAs . hibited proliferation and promoted apoptosis in More and more studies revealed deregulation of glioma cells. Moreover, SOX7 was confirmed to miRNAs in various cancers including lung cancer, be a direct target of miR-616 in glioma cells us- breast cancer, cervical cancer and glioma8-11. Re- ing luciferase assay and Western blotting. Final- cently, several reports showed that deregulation of ly, it was found that down-regulation of miR-616 miR-616 may play important roles in cancer12,13. To or upregulation of SOX7 could suppress the ac- tivity of Wnt/β-catenin signaling in glioma cells. date, the function of miR-616 in glioma and tumor CONCLUSIONS: Our findings indicated that angiogenesis has not been determined. miR-616 acted as a tumor promoter in glioma, Sex-determining region Y-box 7 (SOX7) is and its oncogenic roles were involved in the reg- a putative tumor suppressor in various types of ulation of SOX7 and Wnt/β-catenin signaling. human cancers14. Mechanism assay showed that Moreover, knockdown of miR-616 may provide a SOX7 is a negative regulator of the WNT-β-cat- potential therapeutic strategy for glioma. enin-TCF signaling pathway15. Notably, a number 16 17 Key Words: of miRNAs, including miR‑935 , miR‑452 and miR-616, SOX7, Proliferation, Apoptosis, Wnt/β-cat- miR‑66418, participate in the regulation of SOX7 enin pathway, Glioma. activity in various tissues. However, the associa- tion between miR-616 and SOX7 in glioma cells has not been investigated. Introduction In the present study, we firstly determined the expression pattern of miR-616 in glioma tissues Glioma is the most common type of primary and cell lines. Then, we investigated the role of malignant brain tumor in the central nervous sy- miR‑616 in the regulation of glioma cell prolifera- stem1. Glioma cells showed a high metastatic abi- tion and apoptosis, as well as the underlying me- lity owing to the high proliferation of endothelial chanism involving SOX7 and Wnt signaling.

5630 Corresponding Author: Qing-ling Bai, MM; e-mail: [email protected] MiR-616 regulated SOX7 in glioma

Patients and Methods rer’s instructions. The absorbance at 570 nm was measured by ELISA reader. Patients Glioma tissues and adjacent non‑tumors sam- Flow Cytometry Analysis ples were obtained from 36 Chinese patients (age, Cells were collected and washed twice with 48.2 ± 8.3 years) at Cangzhou Central Hospital phosphate-buffered saline (PBS). 100 μL of this between 2013 and 2015. After resection, samples cell suspension were incubated with 5 μL of FI- were frozen immediately in liquid nitrogen, sto- TC-Annexin V and 5 μL propidium iodide (PI) red at -80°C. None of the patients had received for 15 min in the dark. Apoptotic cells were quan- chemotherapy or radiotherapy prior to surgery. tified using flow cytometry (Beckman-Coulter, Informed consent was obtained from each subject Brea, CA, USA). The experiments were perfor- or subject’s guardian after approval by the appro- med in triplicate. priate hospital Ethics Committee.

Cell Culture and Transfection Western blot Assay Glioma cell lines, including U87 MG, U118 MG, Cells were solubilized in cold radioimmuno- U251, A172 and U87, were obtained from Cell Ban- precipitation assay (RIPA, Biosystems, Foster ks of Shanghai Institutes of Biological Sciences City, CA, USA) lysis buffer (Thermo Fisher (Shanghai, China). A normal human glial cell HA Scientific, Waltham, MA, USA) to extract pro- was purchased from Shanghai Cell Collection (SCC, tein. Total were extracted with RIPA Pudong, Shanghai, China). Cells were cultured in lysis buffer and separated by sodium dodecyl Dulbecco’s Modified Eagle Medium (DMEM) con- sulphate-polyacrylamide gel electrophoresis taining 10% fetal bovine serum (FBS) at 37°C in an (SDS-PAGE); then, they were transferred onto atmosphere containing 5% CO2. the polyvinylidene difluoride (PVDF) mem- The coding region of SOX7 was cloned into brane. The membranes were blocked with 5% pcDNA3.1 plasmid named as pcDNA-SOX7. non-fat dried milk and incubated with primary MiR-616 mimics, mimic control, miR-616 inhi- antibodies. The following antibodies were bitors, inhibitor control were all synthesized by used in this study: SOX7, cyclin D1, β-caten- GenePharma (Pudong, Shanghai, China). Cell in and c-myc. They were purchased from Cell transfection was performed using Lipofectamine Signaling Technology (CST, Danvers, MA, 2000 (Invitrogen, Carlsbad, CA, USA) following USA). GAPDH was purchased from Protein- the manufacturer’s protocol. Tech (Kangchen, Shanghai, China). The pro- tein complexes were detected using enhanced Quantitative Real-time-PCR chemiluminescence (ECL) reagents (Pierce, Total RNA was extracted from tissues and cel- Rockford, IL, USA). ls using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Reverse transcription was performed using HiScript Luciferase Activity Assay II First Strand cDNA Synthesis Kit (Vazyme Biote- In brief, the miR-616-binding site in the SOX7 ch, Qinhuai, Nanjing, China). qRT-PCR assays were 3’-UTR region (wild or mutant-type) was cloned performed on an ABI 7500 fast Real-time PCR sy- downstream of the firefly luciferase gene in a stem (Biosystems, Foster City, CA, USA) according pGL3-promoter vector. Luciferase activities were to the manufacturer’s instructions. The primers for measured by using a dual luciferase assay kit miR-616, SOX7 and GAPDH were purchased from (Promega, Madison, WI, USA). Renilla luciferase Qiagen (Valencia, CA, USA). GAPDH was used as was used for normalization. an internal control. Gene expression was normali- zed to the level of GAPDH within each sample using the relative 2–ΔΔCT method. Statistical Analysis All statistical analyses were performed using Cell Proliferation Assay the SPSS 16.0 software package (SPSS, Chi- Cells were plated in 96-well plates at a densi- cago, IL, USA). Statistical analysis was per- ty of 5×103/well. After the RNA interference as formed with Student’s t-test to compare data above, the proliferation was examined by MTT between two groups. p < 0.05 was considered from days 1 to 5 according to the manufactu- statistically significant.

5631 Q.-L. Bai, C.-W. Hu, X.-R. Wang, J.-X. Shang, G.-F. Yin

Figure 1. The expression of miR-616 was up-regulated in glioma tissues and cell lines. (A) The expression of miR-616 was measured in glioma samples and normal tissues via RT-qPCR. (B) The RT-PCR analysis of miR-616 expression was conducted in HA, U87 MG, U118 MG, U251, U87 and A172 cell lines. *p < 0.05, **p < 0.05.

Results ced the growth of U261 and U87 cells compa- red to control cells (Figure 2C-D). Subsequent- miR-616 Expression is Upregulated in ly, flow cytometry assay showed that miR-616 Human Glioma Tissues and Cell Lines inhibitors transfection was positively correla- To assess the role of miR-616 in glioma, we ted with enhanced apoptosis of U261 and U87 first detected miR-616 expression in tumor tis- cells (Figure 2E-F). We further detected the sue samples and adjacent non-cancerous tissues. expression levels of proliferation-related pro- As shown in Figure 1A, we found that miR- tein Ki67 by Western blot. The results showed 616 expression level was significantly higher that the expression levels of Ki67 in U251 and in glioma tissues compared with adjacent nor- U87 cells transfected with miR-616 inhibitors mal tissues (p < 0.01). Then, we determined the were significantly decreased compared with expression levels of miR-616 in several glioma those in the controls (Figure 2G and 2H). These cell lines. We found that miR-616 was differen- data suggested that miR-616 may play a tumor tially decreased in 5 glioma cell lines compared promoter in glioma. with that in the normal human glial cells (Figure 1B, p < 0.05). Thus, our results indicated that SOX7 is a Direct Target of miR-616 miR-616 may be involved in the progression of in Glioma glioma. TargetScan and miRBase algorithms were em- ployed to search for putative protein-coding gene Down-Regulation of miR-616 Inhibits targets of miR-616. We found that SOX7 may be Glioma cell Proliferation and Induces a potential target (Figure 3A). Then, we perfor- Cell Apoptosis in vitro med dual-luciferase reporter assay to validate our Next, we set out to explore the biological hypothesis. The results showed that miR-616 si- function of miR-616 in glioma progression. We gnificantly suppressed the luciferase activity of reduced miR-616 expression in U251 and U87 the wild-type SOX7 3’-UTR (WT) but not Mut 3’- cells and studied its impacts on proliferation UTR of SOX7 in both U251 and U87 cells (Figure and apoptosis. The results of PCR indicated 3B). Furthermore, in the SOX7 mRNA analysis that miR-616 expression was down-regulated from RT-PCR, miR-616 overexpression decrea- in U261 (Figure 2A, p < 0.01)) and U87 (Fi- sed SOX7 mRNA level (Figure 3C). Besides, in gure 2B, p < 0.01) after transfected with miR- the SOX7 protein analysis from Western blot, 616 inhibitors. Then, MTT and flow cytome- miR-616 overexpression decreased SOX7 protein try assays were performed. We observed that level (Figure 3D). Taken together, SOX7 is a di- down-regulation of miR-616 significantly redu- rect target of miR-616 in glioma cells.

5632 MiR-616 regulated SOX7 in glioma

Figure 2. Down-regulation of miR-616 suppressed proliferation and promoted apoptosis in glioma cells. (A, B) Validation of miR-616 expression levels in U251 or U87 after transfection by qRT-PCR analysis. (C, D) The effect of miR-616 on U251 and U87 cell proliferation was analyzed by MTT assay. (E, F) The effect of miR-616 on U251 and U87 cell apoptosis was analyzed by flow cytometry. (G, H) The expression level of Ki67 in U251 and U87 cells was determined by Western blot. *p < 0.05, **p < 0.05.

MiR-616 and SOX7 were Involved As shown in Figure 4A, we found that down-re- in the Regulation of Wnt/β-Catenin gulation of miR-616 significantly suppressed Wnt Signaling Pathway signaling. On the other hand, over-expression of It has been confirmed that Wnt/β-catenin si- miR-616 also significantly suppressed Wnt signa- gnaling is activated in glioma cell lines. Then, we ling (Figure 4B). These results indicated that miR- examined whether miR-616 and SOX7 affected the 661 contributed to the progression of glioma by tar- expression of the Wnt signaling-related proteins. geting SOX7 via Wnt/β-catenin signaling pathway.

5633 Q.-L. Bai, C.-W. Hu, X.-R. Wang, J.-X. Shang, G.-F. Yin

Discussion silencing miR-616 markedly suppressed the mi- gration and invasion of lung cancer cells by tar- Accumulating evidence has indicated that geting GSK3β. However, miR-616 expression and miR-616 acts as a tumor suppressor in various its effects on glioma are unclear. types of cancer. For instance, Yao et al19 reported In the present study, we showed that miR-616 that the expression levels of miR-616 in gastric was significantly up-regulated in glioma tissues cancer tissues were significantly lower than tho- and cell lines. Then, we investigated the signifi- se in corresponding noncancerous tissues by the cance of miR-616 expression in glioma in vitro miRNA microarray analysis. Zhang et al20 found and found that down-regulation of miR-616 si- that up-regulation of miR-616 was significantly gnificantly suppressed glioma cell proliferation. associated with poor prognosis of hepatocellular Moreover, the results of flow cytometry analysis carcinoma patients. Furthermore, functional as- indicated that down-regulation of miR-616 pro- say indicated that over-expression of miR-616 can moted glioma cell apoptosis. Then, we explored promote the migration, invasion and EMT of he- the effect of miR-616 on the regulation of Ki67 le- patocellular carcinoma cells by targeting PTEN. vels. We found that the expression level of Ki67 in Wang et al21 reported that miR-616 levels were glioma cells transfected with miR-616 inhibitors upregulated in late-stage NSCLCs, and strongly is significantly decreased. Thus, based on these correlated with risk of lung cancer recurrence and results, we supposed that miR-616 may be a tu- metastasis. In vitro and in vivo assay revealed that mor oncogene in glioma.

Figure 3. MiR-616 inhibits SOX7 expression by binding its 3’-UTR. (A) SOX7 was predicted as a target gene of miR-616 by three prediction software packages, and the potential binding site of miR-616 in 3’UTR of SOX7 was presented. (B) The luciferase activity of the wild-type SOX7 3′-UTR (Wt) and mutant SOX7 3’-UTR (Mut) co-transfected with miR-616 mimics or a miR-NC was measured. (C) SOX7 mRNA expression in U251 and U87 cells transfected with miR-616 mimics or miR-NC showed by PCR. (D) SOX7 protein expression in U251 and U87 cells transfected with miR-616 mimics or miR-NC showed by Western blot. *p < 0.05, **p < 0.05.

5634 MiR-616 regulated SOX7 in glioma

Figure 4. The regulation of Wnt pathway by miR-616 and SOX7. (A) Western blot analysis of β-catenin, cyclin D1 and c-myc expression in U251 cells transfected with miR-616 inhibitor or control. (B) Western blot analysis of β-catenin, cyclin D1 and c-myc expression in U251 cells transfected with pnDNA-SOX7 or pcDNA-control. *p < 0.05, **p < 0.05.

The human SOX7 gene is located at the chro- Wnt/β-catenin pathway is one of the most im- mosomal region of 8p23.122. SOX7 plays an im- portant molecular pathways. Moreover, altered portant role in the development and progression of activity of the Wnt/β-catenin signaling pathway various tumors. Many reports showed that SOX7 plays an important role in the development of tu- functioned as a tumor suppressor in primary colo- mors27-28. It has been confirmed that SOX7 was rectal tumors, gastric cancer, prostate cancer and involved in the regulation of Wnt/β-catenin pa- glioma23-26. Recently, several miRNAs have been thway29,30. Thus, we focused on the association reported to exert their effect on tumor progres- between miR-616 and Wnt/β-catenin pathway. We sion by targeting SOX7. For instance, Yang et al16 detected the expression levels of β-catenin and the reported that overexpression of miR-935 promo- downstream of the Wnt/β-catenin signaling ted gastric cancer cell proliferation by targeting pathway. The results indicated that down-regula- SOX7. Bao et al18 found that overexpression of tion of miR-616 suppressed the expression levels miR-664 functioned as a tumor promoter by tar- of the Wnt signaling-related proteins. Moreover, geting SOX7. In our current study, we found that the knockdown of SOX7 also exerted the similar miR-616 negatively regulated SOX7 expression effect. Thus, our findings revealed that miR-616 by targeting its 3’-UTR; also, a forced expression promoted glioma cells proliferation by targeting of miR-616 could suppress the levels of SOX7. SOX7 via Wnt/β-catenin signaling pathway.

5635 Q.-L. Bai, C.-W. Hu, X.-R. Wang, J.-X. Shang, G.-F. Yin

Conclusions 12) Ma S, Chan YP, Kwan PS, Lee TK, Yan M, Tang KH, Ling MT, Vielkind JR, Guan XY, Chan KW. MicroR- NA-616 induces androgen-independent growth of In this study, we firstly report that miR-616 is prostate cancer cells by suppressing expression upregulated in glioma cell lines and tissue sam- of tissue factor pathway inhibitor TFPI-2. Cancer ples, and miR-616 promotes proliferation and Res 2011; 71: 583-592. inhibits apoptosis in glioma cells. We identified 13) Rani S, Gately K, Crown J, O’Byrne K, O’Driscoll L. SOX7 was a targeting gene of miR-616. Further- Global analysis of serum microRNAs as potential more, we also provide evidence to prove that miR- biomarkers for lung adenocarcinoma. Cancer Biol 616 exerts its oncogene role by targeting SOX7 Ther 2013; 14: 1104-1112. via Wnt/β-catenin signaling pathway. Therefore, 14) Stovall DB, Cao P, Sui G. SOX7: from a develop- mental regulator to an emerging tumor suppres- miR-616 may be a future therapeutic target for the sor. 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