YY1 antibodies were obtained from Santa Cruz (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Antibodies specific for p53 (cat. no. 2524) and β-actin (cat. no. 3700) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Goat anti-mouse IgG (cat. no. AP124B) and goat anti-rabbit IgG (cat. no. AP307P) were purchased from EMD Millipore (Billerica, MA, USA).

Human glioma cells U251MG and 293T cells were obtained from the Cell Bank of Shanghai Institutes of Chinese Academy of Sciences (Shanghai, China). Cells were grown at 37°C with 5% CO₂ in DMEM medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% heat-inactivated fetal bovine serum (Evergreen Biological Engineering, Hangzhou, China).

The miR-218 target prediction was performed using bioinformatics algorithms from TargetScan (http://www.targetscan.org/) and PicTar (http://pictar.mdc-berlin.de/).

For overexpression of miR-218, the miR-218 cDNA was inserted into the pGlV3/H1 plasmid (Shanghai GenePharma Co., Ltd., Shanghai, China) using BamHI and MluI sites. For silencing of miR-218, the short-hairpin RNA (shRNA) oligomer (target sequence: GAT CCA CAT GGT TAG ATC AAG CAC AAC GAT ACA TGG TTA GAT CAA GCA CAA ACC GGT ACA TGG TTA GAT CAA GCA CAA TCA CAC ATG GTT AGA TCA A GC ACA ATT TTT TG) was annealed and then subcloned into the pGLV3/H1 plasmid by BamHI and MluI cloning sites. For knockdown of endogenous YY1, a specific shRNA oligomer (target sequence: CCT CCT GAT TAT TCA GAA TAT) was annealed and then introduced into the pLV-shRNA plasmid by BamHI and EcoRI cloning sites (23). For overexpression of YY1, the YY1 cDNA was inserted into the 3X Flag plasmid using EcoRI and XbaI sites. 3X Flag-p53 were obtained from Addgene, Inc. (Cambridge, MA, USA). Cells were transfected with Polyjet (SignaGen Laboratories, Gaithersburg, MD, USA) according to the manufacturer's protocol. The viruses were packaged in 293T cells by cotransfecting the corresponding plasmids with the helper plasmids using Polyjet transfection reagent.

RNA was extracted from stable lines using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following manufacturers' instructions and 1 µg total RNA was subjected for cDNA production using reverse transcription reagents (Roche Diagnostics GmbH, Basel, Switzerland) according to the manufacturer's protocol. RT-qPCR reactions were performed with 3 µl template from each reaction with an ABI 7300 Real-Time PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Green (Roche, Diagnostics GmbH). Primers for the amplification of YY1 and β-actin were as follows: YY1, forward CCT GGC ATT GAC CTC TCA GAT CCA and reverse GGG CAA GCT ATT GTT CTT GGA GCA; β-actin, forward CAT GTA CGT TGC TAT CCA GGC and reverse CGC TCG GTG AGG ATC TTC ATG. The products for YY1 and β-actin were 101 and 195 bp, respectively. PCR reaction mixture (10 µM forward and reverse primers, 10 µl of LightCycler 480 SYBR Green I Master (Roche, Diagnostics GmbH), 3 µl of cDNA template and 6 µl of RNA-free water) was added into the reaction plate, and then run by PCR instrument. The following thermocycling conditions were used for the PCR: Initial denaturation at 95°C for 5 min; followed by 40 cycles of 95°C for 15 sec and 65°C for 35 sec. For each sample, the C_{quantification} cycle (Cq) was determined and normalized to the average of the housekeeping gene (ΔCq=Cq Unknown-Cq Housekeeping gene). The determination of gene transcript levels in each sample was performed using the 2^−ΔΔCq method (24).

The YY1 3′untranslated region (UTR)-Luc reporter was created by ligation of the YY1 3′UTR PCR product into the SacI and XbaI site of the pmirGLO control vector (Shanghai GenePharma Co., Ltd., Shanghai, China). The mutant reporter was generated from pmirGLO-wild-type (WT)-YY1 3′UTR-Luc by deleting the binding site for miR-218 ‘UGAAUGU’. miR-21 promoter-containing (miPPR-21-containing) pmirGLO-basic plasmids were constructed (Shanghai GenePharma Co., Ltd.). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA).

The proliferation of cells was evaluated by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay using an EdU assay kit (Guangzhou RiboBio Co., Ltd., China) according to the manufacturer's protocol. U251MG cell (1×10⁴ cells per well) were incubated in triplicate in a 96-well plate for 48 h and then exposed to 50 µM EdU for additional 2 h at 37°C. Cells were fixed with 4% formaldehyde for 30 min at 25°C and 2 mg/ml glycine was added to neutralize the formaldehyde. Cells were then incubated with 0.5% Triton X-100 for 15 min at 25°C for permeabilization. After washing with PBS three times, the cells were treated with 100 µl 1X Apollo Reaction Cocktail (Guangzhou RiboBio Co., Ltd.) for 30 min. DNA was stained by Hoechst staining for 30 min to determine total number of cells and calculate percentage proliferation. The images were photographed under the Olympus IX-71 inverted microscope (Olympus Corporation, Tokyo, Japan).

A total of 5,000 cells in 100 µl DMEM medium were plated in 96-well plates and grown under normal conditions. After cells had adhered to the plates, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-benzene disulfonate)-2H-tetrazolium monosodium salt (AAT Bioquest, Inc., Sunnyvale, CA, USA) was added into the medium and cells were incubated for 4 h at 37°C. Living cells were counted daily by reading the absorbance at 450 nm using SynergyMx Multi-Mode Microplate Reader (Biotek Instruments, Inc., Winooski, VT, USA).

A total of 200 cells were plated in a 6-cm dish and incubated under normal conditions for 10 days. The cells were fixed with methanol and dyed with 0.05% crystal violet to assess colony staining. Following repeated washing with PBS, images were taken with a camera. Colonies containing more than 50 cells were counted.

Cells were lysed and equal amounts of cell lysates were subjected to 10% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequent to blocking with 5% skimmed milk, the membrane was incubated with primary antibodies [YY1 (1:1,000), β-actin (1:1,000)] at 4°C overnight and then secondary antibodies (1:5,000) at room temperature for 1 h. Bound antibodies were detected by the enhanced chemiluminescence Q6 system (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Band densities were measured using ImageJ software 1.42q (National Institutes of Health, Bethesda, MD, USA). All examined gene expression levels were determined by normalizing the densitometry value of interest to that of the loading control.

SPSS software was used to perform statistical analyses. Data were presented as the mean ± standard error. Statistical analyses were performed using SPSS, version 13.0 (SPSS, Inc., Chicago, IL, USA). Differences in multiple groups were determined by using one-way analysis of variance followed by Student-Newman-Keuls test. Comparison between two groups was performed by Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

Bioinformatics analysis using TargetScan and PicTar algorithms indicated that YY1 is a hypothetical target gene of miR-218 (Fig. 1A). To further confirm the direct interaction between miR-218 and YY1 3′UTRs, plasmids containing WT (pmirGLO-WT-YY1 3′UTR) and mutant (MT)-type (pmirGLO-MT-YY1 3′UTR) YY1 3′UTRs were constructed. Luciferase reporter assays demonstrated that upregulation of miR-218 led to a notable decrease of luciferase activity of pmirGLO-WT-YY1 3′UTR in human glioma cells, without any significant change in luciferase activity of pmirGLO-MT-YY1 3′UTR (Fig. 1B). These results suggest that YY1 may be a direct target of miR-218 in human glioma cells.

Whether YY1 participates in the proliferation of human glioma cells was investigated in vitro by overexpressing shRNA against YY1 to downregulate YY1 expression. The YY1-downregulated stable cell line was estabilshed by lentiviral infection and the infection efficiency was verified through the immunofluorescence images (Fig. 2A). The western blotting assay indicated that knockdown of YY1 expression could increase the expressions of p53 protein (Fig. 2B). Proliferation of glioma cells was examined by EdU, CCK-8 and colony formation experiments. Knocking down YY1 significantly decreased the proportion of EdU positive cells (Fig. 2C and D). CCK-8 experiments demonstrated that knocking down YY1 reduced the proliferation of glioma cells (Fig. 2E). The number of colonies was also reduced upon downregulating YY1 (Fig. 2F and G). Therefore, it was concluded that knocking down YY1 reduces the proliferation of glioma cells.

Several studies have demonstrated that miR-218 has tumor-suppressive functions in human glioma (14,15,18). However, the role of miR-218 in glioma remains unclear. Thus, the current study aimed to investigate the effects of miR-218 on proliferation by regulating YY1 in human glioma cells. miR-218 expression was first knocked down with an miRNA sponge specific for miR-218 (miR-218 sponge). Stable cell lines expressing the miR-218 sponge were constructed in U251MG cell by lentiviral infection (Fig. 3A). The infection efficiency of miR-218 was performed by monitoring the mRNA expression of YY1 (Fig. 3B). CCK-8 experiments demonstrated that knocking down miR-218 accelerated cell proliferation (Fig. 3C). Furthermore, colony formation ability was increased upon downregulation of miR-218 (Fig. 3D and E). Taken together, it was concluded that knocking down miR-218 induced the promotion of proliferation in glioma cells.

To further explore the biological function of miR-218 in glioma cells, miR-218 was upregulated by lentiviral infection (Fig. 4A). The expression of YY1 mRNA was verified through RT-qPCR experiments and was observed to be significantly decreased in response to overexpressed miR-218 (Fig. 4B). Overexpression of miR-218 led to inhibition of glioma cell proliferation, as determined by CCK-8 (Fig. 4C) and colony formation experiments (Fig. 4D and E). Therefore, it was concluded that upregulating miR-218 inhibited the proliferation of human glioma cells.

To clarify the potential mechanism of miR-218 in glioma cells, the YY1 and p53 expression in sponge cells overexpressing miR-218 were detected. Western blot analysis demonstrated that downregulation of miR-218 upregulated the protein level of YY1, promoted the degradation of p53, and when we cotransfected the pLV-shYY1 and miR-218-sponge plasmids into U251MG, the expression levels of p53 were restored compared to upregulating miR-218-sponge (Fig. 5A). The results of EdU experiments and western blot analysis were consistent, and pLV-shYY1 could reverse the effect of miR-218-sponge in cell proliferation (Fig. 5B and C). Upregulating 3X Flag-YY1 and miR-218 led to similar results and the same protein expression trend with overexpressing pLV-shYY1 and miR-218-sponge, as determined by western blotting and EdU (Fig. 5D-F). However, when 3X Flag-p53 was overexpressed in U251MG cell, the expression of YY1 was not detected to be significantly different (Fig. 5G). Therefore, it was inferred that inhibition of glioma cell proliferation by overexpressing miR-218 in glioma cells may be mediated by downregulation of YY1 and upregulation of p53 expression which may be due to the reduction of p53 degradation by YY1.

Together the data indicated that YY1 is a direct target of miR-218 and serves a role in promoting the proliferation of glioma cells. The results suggest that miR-218 suppresses the proliferation of human glioma cells through the YY1/p53 pathway.