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Abstract. – OBJECTIVE: This study is de-signed to investigate the role of miR-363-3p in the cancer development of glioma.

PATIENTS AND METHODS: The expression of miR-363-3p in glioma and adjacent noncan-cerous tissue was measured using quantitative RT-PCR. The expression of a target gene of miR-363-3p, pyruvate dehydrogenase B (PDHB), was determined by Western blot. The level of miR-363-3p was increased or decreased by trans-fected with miR-363-3p mimic or miR-363-3p in-hibitor, respectively. The impact of miR-363-3p on cell growth, apoptosis and invasion was de-termined by CCK-8 (Cell Counting Kit) assay, flow cytometry, and transwell assay. The role of PDHB in mediating the oncogenic activities was demonstrated by co-transfected PDHB vector and miR-363-3p mimic.

RESULTS: Our results have shown that miR-3663-3p level was significantly higher in glioma tissue. Furthermore, miR-363-3p functions as onco-miRNA, promotes cell proliferation, pro-tects against apoptosis, and enhances invasion by directly targeting PDHB.

CONCLUSIONS: MiR-363-3p is an onco-miR-NA, which can be considered as a potential ther-apeutic target in glioma.

Key Words:Glioma, miR-363-3p, Pyruvate dehydrogenase B.

Introduction

GBM (Glioblastoma), accounting for the ma-jority of malignant gliomas, is featured by aggressive growth and invasion1-3. Currently, glioma is treated with temozolomide and ra-diotherapy, which has significantly improved the clinical outcome of patients4. However, the clinical outcomes for patients with glioma are still far from ideal5,6. Therefore, more under-

standing of the factors involved in the patho-genesis and development of glioma will help to develop specific targeting approaches.

MicroRNAs (miRNAs), which belongs to non-coding RNAs (ribonucleic acid) family, have been identified to involve in a variety of cellular processes, such as cell differentiation, prolifer-ation, apoptosis, migration, and invasion7. Mi-croRNAs regulate a plethora of genes expression via binding to 3’ UTR (untranslated regions) of mRNA. Thus, it is leading to the degradation or repression of mRNA translation with a decrease in the particular protein levels8. In the context of glioma, a number of miRNAs have been found to be aberrantly expressed. The miRNAs were ei-ther as oncogene to promote disease progression or as tumor suppressor to inhibit tumor growth9. For instance, miR-451 has been found to nega-tively maintain cell survival and metastasis in glioma10. MiR-221/222 was found to implicate in the progression of glioma by increasing the acti-vation of Akt signaling pathway11. Collectively, these findings evidence the crucial role of miR-NAs in the pathogenesis of glioma.

Pyruvate dehydrogenase B (PDHB), which locates in mitochondria, catalyzes the con-version of Acetyl-CoA from glucose-derived pyruvate12 and serves as the entry point from glycolytic cycle to the citric acid cycle for en-ergy production. Given the crucial role of gly-colysis in the development and progression of human malignancies, the involvement of PDHB in cancer has been reported in many studies13,14. PDHB (pyruvate dehydrogenase B) is found to be aberrantly high expressed in gastric cancer tissue and associated with favorable progno-sis14. However, the role of PDHB in glioma and the underlying regulatory mechanisms, remain elusive.

European Review for Medical and Pharmacological Sciences 2018; 22: 5230-5239

D.-X. XU, J.-J. GUO, G.-Y. ZHU, H.-J. WU, Q.-S. ZHANG, T. CUI

Department of Neurosurgery, The First Affiliated Hospital of Henan University of Science and Technology, Henan, China

Corresponding Author: Tao Cui, MD; e-mail: [email protected]

MiR-363-3p modulates cell growth and invasion in glioma by directly targeting pyruvate dehydrogenase B

MiR-363-3p modulates cell growth and invasion in glioma by directly targeting PDHB

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Patients and Methods

Clinical Specimen Collection and Analysis

The study protocol was reviewed and ap-proved by the Medical Ethical Committee of the First Affiliated Hospital of Univer-sity of Science and Technology of Henan (Luoyang, Henan, China), and all patients signed consent forms before the study. Fresh tumor specimens from patients with glioma and nonneoplastic brains tissues from patients without glioma were collected in the First Affiliated Hospital of University of Science and Technology of Henan (Luoyang, Henan, China). All specimens were examined blindly and independently by 2 senior pathologists. Quantitative RT-PCR (reverse transcription polymerase chain reaction) (qRT-PCR) was used to determine the level of miR-363-3p in tissue samples. Automated capillary Western blot (WES)15) was conducted to determine the level of SOX2 (sex determining region Y-box 2) in tissue samples.

Cell CulturesThe normal human astrocytes (NHA) were

obtained from Cell Bank of Chinese Academy of Science (Wuhan, Hubei, China). The glioma cell lines U87, LV229, U251, LN18 and SHG44 were purchased from Cell Bank of Shanghai Institute of Biotechnology and Cell Biology (Shanghai, China) and incubated in DMEM (Dulbecco mod-ified Eagle medium) Gibco (Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA) without antibiotics at 37°C and 5% CO2.

Cell TransfectionThe miR-363-3p mimics, mimics negative

control (NC), and miR-363-3p inhibitor were obtained from Genepharma (Shanghai, Chi-na). GBM (glioblastoma) cells were transfected with the constructed lentivirus to force expres-sion of miR-363-3p or knockdown miR-363-3p expression.

Quantitative Real-time PCR (qRT-PCR)TRIzol reagent (Tiangen Biotech, Bei-

jing, China) and miRcute miRNA isolation kit (DP501) (Tiangen Biotech, Beijing, China) were used to isolate total RNA and miRNA from cultured cells and tissues, respectively. In short, cDNA (deoxyribonucleic acid) was

synthesized by high capacity cDNA archive kit (Applied Biosystems, Foster City, CA, USA), then qRT-PCR (Polymerase Chain Reaction) was conducted using TaqMan Probe (Applied Biosystems, Foster City, CA, USA) to quantify the level of miR-363-3p, following the manu-facturer’s instructions. U6 was used to normal-ize miR-363-3p level. The sequence of PDHB primer was synthesized as previously de-scribed16. Reverse transcription kit (Promega, Madison, WI, USA) was used to synthesize cD-NA, and then measuring the level of PDHB by RT-PCR with Power SYBR (Synergy Brands) Green PCR Master Mix (Carlsbad, CA, USA) and GAPDH (glyceraldehyde-3-phosphate de-hydrogenase) as a control for normalization. The 2-ΔΔ Ct method was performed to calculate the relative expression of target genes.

Western Blotting AnalysisWestern blotting was performed following

standard protocols. Proteins were detected with specific primary antibodies against PDHB, cleaved caspase-3, cleaved PARP (poly ADP-ri-bose polymerase), and β-actin (Abcam, Cam-bridge, MA, USA). Specific secondary antibod-ies were Goat anti-rabbit IgG-HRP (horseradish peroxidase) (Beyotime, Shanghai, China). Pro-tein levels were measured using a chemilumi-nescent substrate (KPL, Guildford, UK) and visualized using BandScan software (Glyko, Novato, CA, USA).

Luciferase Reporter AssayConstruction predicted miR-363-3p binding

site on PDHB 3’-UTR with psiCHECK-2 dual-lu-ciferase expression vector (Promega, Madison, WI, USA). GBM cells were transfected with pGL3-PDHB-3’UTR-wild-type/mutant and miR-363-3p using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Luciferase activity was detected after 48 hours following transfection, as determined by a dual-luciferase reporter assay system.

Ectopic Expression of PDHBPDHB overexpressing and control vector

were carried out as previously reported17. Over-expression construct, an empty vector was carried into GBM cells by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following manufacturer’s instruction. 48 hours following transfection, Western blotting measured the expression of PDHB.

D.-X. Xu, J.-J. Guo, G.-Y. Zhu, H.-J. Wu, Q.-S. Zhang, T. Cui

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CCK-8 AssayCell proliferation was analyzed using Cell

Counting Kit-8 (CCK-8; Beyotime, Shanghai, China) according to the manufacturer’s instruc-tions. The optical density of viable cells was quantified using a spectrophotometer (BioTek, Winooski, VT, USA).

Flow CytometryApoptosis assay was measured using Apop-

tosis kit (Beyotime, Shanghai, China) following manufacturer’s protocol. Briefly, the cells were harvested at a density of 5x105 cells/mL and incu-bated with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) in the dark for 15 min before detection using a flow cytometer (Millipore, Billerica, MA, USA).

Cell Invasion Assay24-well Transwell coated with Matrigel (BD,

Franklin Lakes, NJ, USA) was carried out to evaluate the invasive ability of glioma cell lines. Briefly, serum-starved cells were re-suspended in DMEM (Dulbecco modified Eagle Medium) containing 1% FBS (fetal bovine serum) and harvested at a density of 2×105 cells/ml. Then, we plated 500 uL of the cell lines to the upper chamber of transwell and filled the lower cham-ber with DMEM containing 10% FBS. After 24-hour incubation, we scarped the matrigel and the non-invasive cell in the upper chamber with cotton swabs, and then fixed invasive cells with 4% formaldehyde and stained with hematoxylin. Five random fields were chosen to count and pho-tograph migrated cells.

Statistical AnalysisData are expressed as the mean ± SD. Statis-

tical comparisons between cell lines and tissues were analyzed by one-way ANOVA followed by Dunnett’s t-test. The comparison of miR-363-3p levels in tumor tissue was performed using stu-dent’s t-test. Experimental data were analyzed with GraphPad Prism software (GraphPad Soft-ware Inc., La Jolla, CA, USA), and p < 0.05 was considered statistically significant.

Results

The Level of miR-363-3p is Aberrantly Higher in Glioma Tissue and Cell Lines

qRT-PCR was performed to examine the ex-pression of miR-363-3p in glioma and non-cancer-

ous tissue was determined using qRT-PCR. The results were presented in Figure 1A. Moreover, we also found that miR-363-3p was significantly up-regulated in glioma cells than noncancerous cells (Figure 1B, p < 0.01).

MiR-363-3p Enhances Cell Growth, Prevents Apoptosis and Promotes Invasion

Transfection of both U87 and U251 cells with miR-363-3p mimic suggested that the levels of miR-363-3p were significantly elevated (Figure 2A). We next investigated whether miR-363-3p affects the cell viability of glioma cell lines, as assessed by CCK-8 assay. Our results showed that ectopic expression miR-363-3p effectively promoted cell proliferation in both U87 and U251

Figure 1. Figure 1. MiR-363-3p is upregulated in glioma tissue and cell lines. A. The level of miR-363-3p was higher in glioma tissue compared with adjacent non tumor tissue. B. The expression of miR-363-3p in glioma cell lines was higher than noncancerous cells. **p < 0.01 vs. control.


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cells. In the following experiments, the role of miR-363-3p on cell apoptosis was evaluated by flow cytometric analysis. Our findings demon-strated that miR-363-3p overexpression effective-ly prevented apoptotic cell death in both U87 and U251 cells (Figure 2C). In addition, the activation of caspase-3 and PARP, which are markers for apoptosis, was also significantly inhibited by miR-363-3p overexpression (Figure 2E). The role of miR-363-3p on the invasiveness of glioma cells was determined by transwell assay. As shown in Figure 2D, miR-363-3p markedly increased the number of cells invaded into the chambers (p < 0.01). Altogether, our findings suggested that miR-363-3p functioned as an oncogene in glioma.

Knockdown of miR-363-3p Suppresses Cell Growth, Induces Apoptosis and Prevents Cell Invasion

To demonstrate the functional role of miR-363-3p as an oncogene, both U87 and U251 cells were transfected with miR-363-3p inhibitor to reduce the expression of miR-363-3p. Figure 3A present-ed that the level of miR-363-3p was significantly decreased in cells after transfection with miR-363-3p inhibitor. Then, we examined whether knockdown of miR-363-3p affected the biological activities of glioma cells.

As shown in Figure 3B, miR-363-3p inhibitor markedly resulted in the loss of cell viability. In addition, miR-363-3p inhibitor led to significant increase in apoptotic cell death (Figure 3C). The anti-apoptotic role of miR-363-3p was also demonstrated by that miR-363-3p inhibitor effec-tively lowered the level of activated caspase-3 and PARP (Figure 3E). Moreover, miR-363-3p inhibi-tor markedly repressed the invasive ability of gli-oma cells. Collectively, our findings revealed that miR-363-3p functioned as an oncogene in glioma.

PDHB is a Direct Target by miR-363-3pTo further demonstrate the role of miR-363-

3p in the development of glioma, we utilized bioinformatics database, MicroCosm Target search for the potential targets of miR-363-3p. Based on our searching results, we postulated that PDHB might be a target for miR-363-3p, responsible for the oncogenic effect of miR-363-3p in glioma (Figure 4A). To validate our suggestion, we constructed luciferase vector and conducted luciferase activity assay. As shown in Figure 4B, luciferase activity of the reporter containing wild-type 3’-UTR of PDHB was markedly reduced in cells transfected with

miR-363-3p mimic while no significant change in luciferase activity of the reporter containing mutated 3’-UTR was observed, which indicat-ed that PDHB was directly targeted by miR-363-3p. Moreover, results also presented that miR-363-3p was inversely associated with the expression of PDHB in glioma cell (Figure 4C and 4D). To further explore the relation-ship between PDHB and miR-363-3p, we also examined the expression of PDHB on mRNA and protein levels in tissues. Results presented that expression of PDHB on mRNA and protein levels were both correlated with miR-363-3p level in glioma tissue (Figure 4E and 4F). In summary, these findings supported that miR-363-3p modulated the expression of PDHB both at transcription and translation level.

Ectopic Expression of PDHB Compromises the Oncogenic Effect of miR-363-3p in Glioma Cells

miR-363-3p mimic promoted cell growth and was attenuated by ectopic expression of PDHB (Figure 5). The protecting effect of miR-363-3p on cell apoptosis in both cells was also sig-nificantly abolished by PDHB overexpression (Figure 5B). Furthermore, the enhancing effect of miR-363-3p on cell invasion was also com-pletely abolished by transfection with PDHB overexpressing vector (Figure 5C). Altogether, our experimental results showed that PDHB mediated the oncogenic activities of miR-363-3p in glioma cells.

Discussion

Since the end of the last century, the role of miRNAs in tumorigenicity and tumor progres-sion has attracted the interest of researchers18. Through modulating different target genes by binding the 3′-UTR portion of mRNAs through translational repression or degradation19. miR-NAs have been found to serve as either oncogenes or tumor suppressors in a plethora of human malignancies. Numerous20-22 studies suggested that the miRNAs are involved in the apoptosis, proliferation, invasion, migration of cancer cells and stem cell differentiation. In 2013, Sun et al23 have demonstrated that podoplanin dysregulation caused by down-regulation of miR-363-3p con-tributes to invasion and metastasis of head and neck squamous cell carcinoma. Qiao et al24 have identified that miR-363-3p modulated gastrin-re-


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leasing peptide receptor-mediated tumorigenicity and promoted metastasis in neuroblastoma. In he-patocellular carcinoma, it has been reported that miR-363-3p exerted its anti-growth effect via di-rectly targeting specificity protein 125. In colorec-tal cancer, SOX4 has been advanced to be a direct target and accounting for the inhibitory effect of miR-363-3p on the epithelial-to-mesenchymal transition (EMT) and metastasis26. Conversely, the oncogenic role of miR-363-3p has also been evidenced. Hsu et al27 has identified the critical roles for miR-363-3p in the increment of gas-tric carcinogenesis via targeting c-Myc promoter binding protein 1. Zhang et al28 also showed that miR-363-3p was aberrantly higher expressed in gastric cancer tissues and regarded as an indepen-dent prognostic marker for postoperative recur-

rence and lower overall survival. In addition, they also found F-box and WD-repeat domain-con-taining 7 were the novel targets by miR-363-3p in gastric cancer28. In prostate cancer, increase in miR-363-3p expression positively regulated cell proliferation and promoted EMT29. In terms of glioma, it has been observed that miR-363-3p maintains human glioblastoma stem cell survival through direct targeting caspase-3, caspase-9, and Bim30. In line with their study, we demonstrated that miR-363-3p expression was higher in glioma tissue compared with adjacent non-tumor tissue. Furthermore, enforced expression of miR-363-3p significantly enhanced cell growth, protected against apoptosis, and promoted cell invasion, which provides further evidence to support the oncogenic role of miR-363-3p in glioma.

Figure 4. MiR-363-3p directly targets PDHB. A. Schematic illustration of the putative binding sites between wild-type or mutant 3’-UTR of PDHB and miR-363-3p. B. Luciferase activity of construct of wild-type PDHB is decreased by miR-363-3p in both U87 and U251 cells. C. MiR-363-3p mimic decreases the expression of PDHB mRNA. D. MiR-363-3p mimic decreases the expression of PDHB protein. E. MiR-363-3p in glioma tissue inversely correlates with mRNA of PDHB. F. MiR-363-3p in glioma tissue inversely correlates with protein of PDHB. ** p < 0.01 vs. control.


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In the current work, we identified PDHB as a novel target responsible for the onco-genic effect of miR-363-3p in glioma. As a mitochondrial enzyme, PDHB catalyzes the transformation of pyruvate, which is gener-ated by glucose metabolism to Acetyl-CoA12. By modulating this metabolic pathway, PDHB functions like a switch to glycolytic pathway, which is crucial for energy production in can-cerous cells. In fact, accumulating evidence has shown the critical role of PDHB in cancer progression and metastasis13,14. The value of PDHB as an independent prognostic marker

and predictor for favorable clinical outcomes, has been documented in gastric cancer14. sug-gesting that PDHB may function as a tumor suppressor. Recently, miR-203, miR-146b-5p and miR-370 have been identified to serve as an oncogene by repressing the expression of PDHB in ovarian cancer, colorectal cancer and melanoma, respectively17,31,32. The results presented in the current study also demon-strated that suppression of PDHB mediated the oncogenic effect of miR-363-3 in glioma, which indirectly validated the role of PDHB as a tumor suppressor.

Figure 5. PDHB overexpression attenuates the oncogenic activity of miR-363-3p in glioma cells. A. Increase of PDHB expression compromises the promoting effect of miR-363-3p on cell growth. B. PDHB overexpression abolishes the protecting effect of miR-363-3p on apoptosis. C. PDHB overexpression abrogates the enhancing effect of miR-363-3p on cell invasion. **p < 0.01 vs. control, ^^p < 0.01 vs. miR-363-3p.


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Conclusions

We showed that miR-3663-3p level was mark-edly higher in glioma tissue. Furthermore, miR-363-3p serves as an onco-miRNA and promotes cell proliferation, protects against apoptosis and enhances invasion by directly targeting PDHB.

Conflict of InterestThe Authors declare that they have no conflict of interests.

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MiR-363-3p modulates cell growth and invasion in glioma by ...€¦ · MiR-363-3p modulates cell growth and invasion in glioma by directly targeting PDHB 5233 Figure 2. Increase in