Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), TRIzol reagent, the TaqMan MicroRNA assay kit, the bicinchoninic acid (BCA) protein assay kit and Lipofectamine^® 2000 were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). The demethylation drug 5-Aza-2′-deoxycytidine (Aza) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The miRNeasy mini kit was purchased from Qiagen (Valencia, CA, USA). Mouse monoclonal anti-E-cadherin (1:500; cat. no. YM0208), mouse monoclonal Vimentin (1:500; cat. no. YM0645), rabbit polyclonal Caspase 3 (1:500; cat. no. YT0656), mouse monoclonal BH3 interacting-domain death agonist (Bid; 1:500; cat. no. YM0062), rabbit polyclonal phosphorylated B-cell lymphoma-2 (Bcl-2; 1:500; cat. no. YP0031) and mouse monoclonal β-actin (1:500; cat. no. YM3028) antibodies were purchased from ImmunoWay (Cambridge, UK). Goat anti-mouse (1:1,000; cat. no. SA001) and goat anti-rabbit (1:1,000; cat. no. SA009) IgG(H+L)-HRP secondary antibodies were purchased from Auragene Bioscience (Changsha, China). A cell invasion assay kit was purchased from Merck Millipore (Darmstadt, Germany).

All protocols in the present study were approved by the Ethics Committee of Central South University (Changsha, China). Written informed consent was obtained from all patients with ccRCC included in the study. A total of six ccRCC tissues and matched adjacent normal tissues were collected at the Department of Nephrology, Xiangya Hospital of Central South University. None of the patients had received a blood transfusion, radiotherapy or chemotherapy prior to the surgery. All samples were immediately snap-frozen in liquid nitrogen (Auragene Bioscience) following surgical removal and stored at −80°C until further use.

The present study utilized five human ccRCC cell lines, 786-O, ACHN, SN12C, A704 and TK10, as well as a normal renal cell line, HEK293, which were obtained from the Cell Bank of Central South University. Cells were cultured in DMEM with 10% FBS in a humidified atmosphere containing 5% CO₂ at 37°C.

miRNAs were isolated from tissues or cells using the miRNeasy mini kit according to the manufacturer’s instructions. The miRNA expression was then determined via reverse transcription quantitative polymerase chain reaction (RT-qPCR) using the TaqMan MicroRNA assay kit on a 7500 Fast Real Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA), in accordance with the manufacturer’s instructions. U6 was used as an endogenous control. For each sample, three independent experiments were performed. The relative expression levels of mRNA and miRNA were analyzed using the 2^−∆∆Ct method (17).

Genomic DNA was extracted from ccRCC 786-O and ACHN cells using a genomic DNA extraction kit (Takara, Dalian, China). Genomic DNA (1 μg) was modified with bisulfite using the Epitect bisulfite kit (Qiagen) according to the manufacturer’s instructions, and eluted in a total of 40 ml elution buffer (Auragene Bioscience). Subsequently, 2 ml modified DNA was used as the template for the BSP reaction. The primer sequences were as follows: miR-429 forward, 5′-GTGACCTGGCTCCAGGAAAGGC-3′, and reverse, 5′-CAGATGGAAAAGATGAAACAATGGG-3′. The PCR cycling conditions were set at: 94°C for 4 min, followed by 35 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 5 min, and a final step at 72°C for 5 min. The PCR products were gel purified, cloned into the pUC18-T plasmid (Sangon Biotech, Shanghai, China) and subsequently sequenced by BGI (Wuhan, China).

Genomic DNA was extracted using a genomic DNA extraction kit (Takara). Genomic DNA (1 μg) was modified with bisulfite using the Epitect Bisulfite kit (Qiagen), according to the manufacturer’s instructions and eluted in a total of 40 ml elution buffer. MS-PCR was performed on bisulfate-treated DNA. The primer sequences were as follows: Methylated miR-492 forward, 5′-CGGGGATATTATCGAGGTATATTC-3′ and reverse, 5′-AACTAACACAAACCCTCTACCG-3′ and unmethylated miR-492 forward, 5′-TGGGGATATTATTGAG GTATATTTG-3′ and reverse, 5′-AAACTAACACAAACC CTCTACCAC-3′. The PCR cycling conditions were set at: 94°C for 4 min, followed by 35 cycles at 94°C for 30 sec, 55°C for 30 sec, 72°C for 5 min, and a final step at 72°C for 5 min.

Western blotting was used to examine the protein expression levels in each group. Cells were lysed in cold radioimmunoprecipitation buffer (Auragene Bioscience). The BCA Protein assay kit was used to determine the protein concentration and was used in accordance with the manufacturer’s instructions. Subsequently, the proteins were separated by 10% SDS-PAGE (Auragene Bioscience) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Auragene Bioscience). The PVDF membrane was blocked in 5% nonfat dried milk in phosphate-buffered saline (PBS) for 4 h. Subsequently, the PVDF membrane was incubated with specific primary antibodies at 37°C for 3 h. Following three washes in PBS, each for 5 min, the PVDF membrane was incubated with the appropriate secondary antibody at 37°C for 1 h. Following a further three washes in PBS, each for 5 min, an enhanced chemiluminescence western blotting kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to detect the immune complexes on the PVDF membrane.

The human ccRCC cell lines, 786-O and ACHN were treated with Aza (15.55 nM), or PBA (1.5 nM) or both in combination, with or without anti-miR-429 (20 μM) (HmiR-AN0497; Genecopoeia, Guangzhou, China), for 72 h.

CCK-8 was used to evaluate cell proliferation. A total of 5×10³ cells were seeded in 96-well plates for 24 h, treated with the indicated drugs and further incubated for 0, 24, 48 and 72 h, respectively. At 1 h prior to the completion of the incubation, 10 μl CCK-8 was added to each well. The optical density at 450 nm in each well was determined using an enzyme immunoassay analyzer (Multiskan M3; Thermo Fisher Scientific).

For all groups, 10×10³ cells/well were plated in a 96-well plate and incubated for 0, 24, 48 and 72 h, respectively, at 37°C with 5% CO₂. To assess cell proliferation, 50 μl MTT (5 mg/ml; Auragene Bioscience) in PBS was added and cells were then incubated for 4 h at 37°C and 5% CO₂. Subsequently, the supernatant was removed and 150 μl dimethyl sulfoxide (Auragene Bioscience) was added. The absorbance was detected at 450 nm with a Microplate Reader (Model 680 XR; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Flow cytometry was used to determine the level of cell apoptosis. At 24 h post-transfection, cells were ha r vested a nd washed twice with cold PBS. Subsequently, 10×10⁵ cells were resuspended in 200 μl binding buffer with 10 μl Annexin-V-fluorescein isothiocyanate and 5 μl propidium iodide-phycoerythrin and incubated in the dark for 30 min. Following this stage, 300 μl binding buffer (Keygentec Biotech. Co., Ltd., Nanjing, China) was added and cells were subjected to flow cytometric analysis (Moflo XDP; Beckman Coulter, Krefeld, Germany).

Cells were administered the indicated drug treatments for 72 h, starved in serum-free medium for 24 h and then resuspended in serum-free medium. The cells were added to the upper chamber of a transwell (Transwell kit; BD Biosciences, Bedford, MA, USA), while the lower chamber was filled with base medium containing 10% FBS. Following incubation for 24 h, cells attached to the bottom of the chamber were stained with crystal violet (Auragene Bioscience) for 20 min and then washed and air-dried. Invasive cells were observed under a microscope (AE31; Motic, Fujian, China).

Cells administered with the indicated drug treatments for 72 h were seeded into a 24-well plate and 500 μl fibronectin (20 μg/ml; Sigma-Aldrich) was added into each well. The 24-well plate was incubated at 4°C overnight. Following washing with PBS, 1% bovine serum albumin (Auragene Bioscience) was added to each well for blocking at 37°C for 2 h. Subsequently, the blocking solution was removed and the cells were dried. Cells were then digested and centrifuged at 200 × g for 5 min. Serum-free medium (HyClone, Beijing, China) was used to produce a single-cell suspension and the cell concentration was adjusted to 10⁶ cells/ml. The cell suspension (1 ml) was added into each well with a slide (Auragene Bioscience) and then incubated at 37°C with 5% CO₂ for 2 h. Following the removal of non-adherent cells using PBS, 4% neutral formalin (Auragene Bioscience) was used to fix adherent cells for 3 h and each well was treated with 500 μl crystal violet and incubated for 2 h. Adherent cells were then observed under a microscope (AE31; Motic).

Data are expressed as the mean ± standard deviation of three independent experiments and were analyzed using SPSS 17.0 statistical software (SPSS, Inc., Chicago, IL, USA). The differences between groups were determined using a one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of miR-492 in ccRCC tissues and cells, as well as in their matched adjacent tissues and normal renal cells were examined. As shown in Fig. 1A, the expression of miR-492 was significantly reduced in ccRCC tissues compared with that of their matched adjacent tissues. In addition, analogous results were observed in the ccRCC cell lines, 786-O, ACHN, SN12C, A704 and TK10, compared with HEK293 cells. The methylation status in the CpG island of the miR-492 promoter, which may be involved in the downregulation of miR-492, was further investigated. MS-PCR was used to determine the methylation status in the CpG island of the miR-492 promoter in six ccRCC tissues, as well as two normal adjacent tissues. As shown in Fig. 1B, tumor 1 and tumor 4 exhibited hemimethylation and tumors 2, 3, 5 and 6 exhibited methylation. Conversely, the two normal adjacent tissues were observed to be unmethylated. These findings suggested that hypermethylation of the miR-492 promoter may contribute to the downregulation of miR-492 in ccRCC.

Following treatment of the ccRCC cell lines, 786-O and ACHN, with Aza, PBA or Aza + PBA, the methylation status of the CpG island of the miR-492 promoter was determined using BSP. As shown in Fig. 2A, a significantly higher level of methylation was observed in the 786-O and ACHN cells following treatment with Aza, PBA or Aza + PBA. In addition, the expression levels of miR-492 in each group were determined. As indicated in Fig. 2B, miR-492 expression was significantly upregulated following treatment with Aza and PBA, particularly following treatment with a combination of Aza + PBA.

The effects of miR-492 upregulation on ccRCC cells were further investigated. As shown in Fig. 3A, the epigenetic drug-induced upregulation of miR-492 significantly inhibited the proliferation of ccRCC cells. To further confirm these findings, ccRCC cells were transfected with anti-miR-492, which is able to reverse the upregulation of miR-492 induced by treatment with Aza + PBA, and further MTT investigation revealed that cellular proliferation was enhanced in the Aza + PBA + anti-miR-492 group, when compared with that in the Aza + PBA group (Fig. 3B). These data suggested that miR-492 has a suppressive role in the regulation of ccRCC cell proliferation.

The effect of miR-492 on apoptosis was determined in the ccRCC cell lines, 786-O and ACHN. As shown in Fig. 4, the upregulation of miR-492 induced by treatment with Aza + PBA significantly promoted apoptosis in the ccRCC cell lines, 786-O and ACHN. However, transfection with anti-miR-492 markedly attenuated this effect. These findings suggested that miR-492 upregulation was able to induce ccRCC cell apoptosis.

As shown in Fig. 5A, the upregulation of miR-492 induced by epigenetic drug treatment significantly inhibited the invasion of the ccRCC cell lines, 786-O and ACHN, which was attenuated by transfection with anti-miR-492. Furthermore, miR-492 upregulation induced by treatment with Aza + PBA markedly promoted cell adhesion in the ccRCC cell lines, 786-O and ACHN, which was also inhibited by transfection with anti-miR-492 (Fig. 5B). These findings suggested that miR-492 upregulation was able to inhibit invasion while promoting adhesion in ccRCC cells.

To further investigate the molecular mechanism underlying the effects of miR-492 in ccRCC cells, the expression of several key factors associated with cell survival and adhesion in ccRCC cells were examined following treatment with epigenetic drugs. As shown in Fig. 6, the upregulation of miR-492 expression induced by treatment with Aza + PBA promoted the expression of pro-adhesive E-cadherin, and inhibited the expression of anti-adhesive Vimentin, consistent with the aforementioned data demonstrating that miR-492 upregulation promoted adhesion in ccRCC cells. In addition, upregulation of miR-492 also inhibited the expression of the anti-apoptotic protein Bcl-2, and enhanced the expression of pro-apoptotic Bid and Caspase 3, which was consistent with the findings that miR-492 upregulation promoted ccRCC cell apoptosis.