Fresh human glioma and adjacent non-cancerous tissues were obtained from 60 patients who underwent therapeutic removal of gliomas at Taihe Hospital, Hubei University of Medicine. Patients did not receive any chemotherapy or radiotherapy before surgical treatment. All clinical specimens were collected and used after obtaining informed consent from each patient enrolled in the present study. All specimens were stored in liquid nitrogen for further investigation. The protocol which involved clinical specimens in the present study was approved by the Research Ethics Committee of Hubei University of Medicine.

Human glioma cell lines including U87 and U251, and human kidney epithelial cell line (293T) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). These cells were cultured in Dulbeccos modified Eagles medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 mg/ml streptomycin. All cell cultures were maintained in a humidified cell incubator with 5% CO₂ at 37̊C.

miR-520c and negative control (NC) mimics, miR-520c and NC inhibitors (anti-NC) were obtained from Genecopoeia (Guangzhou, China), and were then transfected into glioma cells with Lipofectamine 2000 following the manufacturers protocol. Retroviral vectors pMMP-TGFBRII were generated by inserting the TGFBRII cDNA into pMMP. Retrovirus packaging and transduction were previously described (16).

Total RNA from glioma cells was extracted by miRNeasy Mini kit (Qiagen, Hilden, Germany) and total RNA from glioma tissues was extracted with TRIzol reagent (Ambion, Thermo Scientific, Shanghai, China). miR-520c levels in these samples were assayed using TaqMan MicroRNA assays based on the manufacturer's protocol (Applied Biosystems, Carlsbad, CA, USA). Real-time PCR of TGFBRII was performed using an UltraSYBR Mixture (CW0957; CWBIO, Beijing, China) and an LC480 PCR System (Roche, Indianapolis, IN, USA). The primers for miR-520c and U6, TGFBRII and GAPDH were obtained from Genecopoeia. The expression levels of mRNA and miR-520c were normalized to the endogenous controls GAPDH and U6 respectively, using the 2^−ΔΔCt method.

To investigate whether miR-520c interacted with the 3′-UTRs of TGFBRII, wild-type (wt) 3′-UTR of TGFBRII predicted to interact with miR-520c was amplified. Then, the wt 3′-UTR of TGFBRII and the corresponding vectors (NC, miR-520c mimic, anti-NC, miR-520c inhibitor or mutant miR-520c) were co-transfected into 293T cells by Lipofectamine 2000. Forty-eight hours after co-transfection, the cells were lysed and assayed using Dual-Luciferase^® Reporter Assay kit (Promega, Madison, WI, USA) based on the manufacturer's instructions.

Glioma cells transfected with the corresponding vectors were seeded in 6-well plates to form a single confluent cell layer. Wounds were made in the confluent cell layer using 100-µl tips. Following wound scratching (0 and 12 h), the width of the wounds were photographed with a phase-contrast microscope.

The migratory and invasive abilities of glioma cells were evaluated with Transwell chambers (BD Biosciences, Franklin Lakes, NJ, USA). Glioma cells (5–10×10⁴) suspended in 100 µl serum-free medium were seeded into the upper chamber, and the lower chamber was filled with 20% FBS to induce glioma cell migration or invasion through the membrane. Matrigel (1:6 dilution; Becton-Dickinson Labware, Bedford, MA, USA) was added to the upper chamber for the invasion assay. Twenty-four hours later, cells that migrated or invaded the Transwell membrane were stained with crystal violet and the number of cells was counted under a microscope. TGF-β1 (2.5 ng/ml; R&D Systems, Minneapolis, MN, USA) was used for inducing glioma cell migration and invasion.

Before protein extraction, glioma cells were washed with phosphate-buffered saline (PBS) to remove the culture media. Cellular proteins were obtained from glioma cells using RIPA lysis buffer and the protein concentrations were assessed using the BSA method. Cellular proteins (30 µg) were separated by 10% SDS-PAGE, and were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk at room temperature for 1 h, the membranes were probed with primary antibodies at 4̊C overnight. Then, the membranes were incubated with the corresponding secondary antibodies at room temperature for 2 h. The primary antibodies used in the present study included: TGFBRII (K105) (#3713; Cell Signaling Technology, Danvers, MA, USA), CD44 (WL0140; Wanleibio, Shanghai, China) and GAPDH (G8140; US Biological, Swampscott, MA, USA).

Before IHC staining, glioma tissues were fixed with 4% formalin and embedded with paraffin. Then, the embedded tissues were cut into 4-µm thick sections and visualized by IHC staining following the standard protocol in order to evaluate the expression level of TGFBRII (Cell Signaling Technology) in glioma tissues.

All quantitative data are presented as the mean ± the standard error of the mean (SEM). Statistical analyses including Pearson Chi-squared test, a two-tailed Student's t-test, Kaplan-Meier method, the log-rank test and Spearman's correlation analysis were performed with GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). P<0.05 was considered to indicate a statistically significant result.

To examine the expression status of miR-520c in glioma, qRT-PCR was performed for 60 pairs of glioma and adjacent non-tumor tissues. Our results showed that glioma tissues had significant decreased expression levels of miR-520c compared with adjacent non-tumor tissues (P<0.05; Fig. 1A). To clarify the clinical significance and prognostic value of miR-520c in glioma, all patients were divided into two groups (miR-520c low-expressing and miR-520c high-expressing groups) according to the median level of miR-520c expression. As shown in Table I, patients with low expression of miR-520c had advanced World Health Organization (WHO) stage (P=0.024). Furthermore, Kaplan-Meier analysis showed that patients with low expression of miR-520c exhibited a significantly decreased overall survival rate (P<0.001; Fig. 1B). These results indicate that miR-520c probably plays an oncogenic role in glioma.

Next, we explored whether miR-520c modulates the migration and invasion of glioma cells. Transfection of miR-520c mimic into U251 cells significantly increased the expression level of miR-520c (P<0.05; Fig. 2A). The wound healing assays showed that the migration of U251 cells was significantly decreased after miR-520c overexpression (P<0.05; Fig. 2B). In addition, Transwell assays demonstrated that overexpression of miR-520c prominently suppressed the migration and invasion of U251 cells (P<0.05, respectively; Fig. 2C). In contrast, miR-520c inhibitor significantly decreased the expression level of miR-520c in U87 cells (P<0.05; Fig. 3A). Subsequently, miR-520c silencing significantly facilitated the migration and invasion of U87 cells (P<0.05, respectively; Fig. 3B and C). Thus, miR-520c reversed the migratory and invasive abilities of the glioma cells.

To disclose the underlying molecular mechanisms of the biological function of miR-520c in glioma cells, TargetScanHuman 7.1 (http://www.targetscan.org) was used to search for the downstream target of miR-520c. TGFBRII, a critical regulator of the TGF-β pathway in glioma (17), was recognized as a potential downstream target of miR-520c. U251 cells that were transduced with corresponding vectors were subjected to immunoblotting for TGFBRII and CD44, a known downstream target of miR-520c (14). Notably, miR-520c overexpression decreased the levels of TGFBRII and CD44 protein, while miR-520c silencing increased the expression of these proteins (Fig. 4A). Furthermore, qRT-PCR also confirmed that miR-520c overexpression downregulated the mRNA levels of CD44 and TGFBRII (P<0.05; Fig. 4B). However, miR-520c knockdown showed no significant effect on mRNA expression (Fig. 4B). As shown in Fig. 4C, the 3′-UTR of TGFBRII contained two putative binding sites for miR-520c. Subsequently, we performed luciferase assay to investigate whether miR-520c could bind to the putative binding sites in the 3′-UTR of TGFBRII. Alteration of miR-520c inversely regulated the luciferase activity of TGFBRII 3′-UTR (P<0.05; Fig. 4D), while mutant miR-520c did not exhibit any influence on the luciferase activity of TGFBRII 3′-UTR (Fig. 4D). Therefore, these data indicate that TGFBRII is a direct downstream target of miR-520c in glioma.

Glioma and non-tumor tissues were subjected to qRT-PCR for TGFBRII mRNA. Our data indicated that the expression levels of TGFBRII mRNA in glioma tissues were significantly higher than those in adjacent non-cancerous tissues (P<0.05; Fig. 5A). Spearman's correlation analysis indicated that miR-520c was strongly correlated with TGFBRII mRNA expression in the glioma specimens (r=−0.910, P<0.001; Fig. 5B). Moreover, immunohistochemical staining indicated that miR-520c high-expressing tumors showed weak staining of TGFBRII, while miR-520c low-expressing tumors showed strong staining of TGFBRII (P<0.05; Fig. 5C and D).

Next, U251 cells with miR-520c overexpression were infected with empty vector (EV) or TGFBRII retroviruses. Restoration of TGFBRII expression was confirmed by western blotting (P<0.05; Fig. 6A). Wound healing and Transwell assays indicated that TGFBRII restoration abrogated the effects of miR-520c overexpression in U251 cells with increased migratory and invasive abilities of glioma cells (P<0.05, respectively; Fig. 6B and C). Since TGFBRII is a target of miR-520c, and TGFBRII is an important component in TGF-β1-induced migration and invasion, miR-520c-overexpressing U251 cells were treated with TGF-β1 (2 µg/ml). As expected, the induction of TGF-β1 showed no effect on miR-520c-overexpressing U251 cells (Fig. 7). Collectively, TGBRII is not only a target but also a functional mediator of miR-520c.