A total of 10 ccRCC tissue samples and adjacent tissue samples were harvested from patients at the Second Xiangya Hospital (Changsha, China) between July 2013 and May 2014. The patients included 5 females with an average age of 57 years old, and 5 males with an average age of 64 years old. The present study was approved by the ethics committee of the Second Xiangya Hospital, and written informed consent was obtained from the patients.

TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to isolate total RNA from the snap frozen tissues. The isolated RNA was treated with DNase I (Invitrogen; Thermo Fisher Scientific, Inc.). The ratio of 28S/18S was analyzed by Glyko Bandscan 5.0 (Beijing Sage Creation Science Co., Ltd., Beijing, China). RNA quality and quantity were determined spectrophotometrically (Shanghai Spectrum Instruments Co., Ltd., Shanghai, China) at 260 and 280 nm, respectively. RT-qPCR was performed as previously described (16,17) using Roche Molecular Light Cycler (Roche Diagnostics, Mannheim, Germany). The specific reaction primers were as follows: β-actin forward, 5′-AGGGGCCGGACTCGTCATACT-3′, and reverse, 5′-GGCGGCACCACCATGTACCCT-3′. The specific primers sets for miR-21 and U6 and the PCR mix were purchased from GeneCopoeia, Inc. (Rockville, MD, USA). The expression levels of U6 were used as an endogenous control. The reaction for each sample was performed in triplicate. Relative expression levels were calculated using the 2^−∆∆Ct method (18).

To investigate EMT marker protein expression levels in cells, western blotting was performed. Protein concentrations were determined using a Bradford protein assay kit (Auragene Biosciences, Changsha, China). Protein (100 µg) from each sample was loaded onto a 7.5% polyacrylamide gel (Amresco, LLC, Solon, OH, USA). Following electrophoresis, the SDS-PAGE separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk in phosphate-buffered saline (PBS) and then incubated at room temperature for 3 h with the following primary antibodies: Monoclonal mouse anti-human fibronectin (cat. no. sc-271098; 1:500), polyclonal rabbit anti-human N-cadherin (cat. no. sc-7939; 1:200), polyclonal rabbit anti-human vimentin (cat. no. sc-5565; 1:500), polyclonal rabbit anti-human E-cadherin (cat. no. sc-7870; 1:1,000) purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), or with monoclonal rabbit anti-human β-actin (cat. no. ab129348; 1:2,000; Abcam, Cambridge, MA, USA) in PBS for 3 h. The membrane was then reprobed with polyclonal goat anti-mouse (ab6789; 1:5,000) and goat anti-rabbit (ab97051; 1:10,000) secondary antibodies (Abcam) conjugated with horseradish peroxidase for 40 min at room temperature. Blots were processed using an enhanced chemiluminescence kit (Santa Cruz Biotechnology Inc.) and exposed to film (Kodak, Rochester, NY, USA).

The 786-O and UOK-117 ccRCC cells were obtained from RIKEN BioResource Center (Ibaraki, Japan). Cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, GA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C with 5% CO₂. HK-2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured as a monolayer in Keratinocyte Serum Free Medium (K-SFM) supplemented with 0.05 mg/ml bovine pituitary extract, 5 ng/ml human recombinant epidermal growth factor (EGF; Invitrogen; Thermo Fisher Scientific, Inc.) and 10% fetal bovine serum, 50 mg/ml penicillin and 50 mg/ml streptomycin (all from Invitrogen; Thermo Fisher Scientific, Inc.).

The miRNA inhibitors 2′-O-methyl antisense oligonucleotide targeting miR-21 (anti-miR-21), the miRNA inhibitor negative control (anti-miR-C), the pre-miR-21, and the mimic positive control were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China).

For transient transfection, a complex of Lipofectamine 2000 (Invitrogen Life Technologies) and plasmids or RNA oligonucleotides was prepared according to the manufacturer's instructions and directly combined with cells in 24-well cell culture plates at a density of 3×10⁴ cells per well.

Cells were seeded at a density of 10,000 cells per well into 96-well culture plates, one day after transfection with lentivirus. Cells were further cultured for another 6, 24, 48 and 72 h. To assess cell proliferation, cells were incubated with 20 µl MTT at a final concentration of 0.5 mg/ml for 4 h at 37°C. After removing the culture supernatant, 150 µl dimethyl sulfoxide (DMSO, Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China) was added to solubilize the crystals for 20 min at room temperature. Absorbance was measured at a wavelength of 492 nm with a fluorescence plate reader (Molecular Devices, Sunnyvale, CA, USA).

Apoptosis of ccRCC cells was induced by serum deprivation and cycloheximide (CHX; Sigma-Aldrich, St. Louis, MO, USA) treatment. UOK-117 and 786-O cells were pre-transfected with pre-miR-21 or anti-miR-21 and corresponding controls (50 nM), and then 24 h after transfection, cells were exposed to CHX. Following a 4-h incubation with CHX at 37°C, the cells were digested with trypsin (Beyotime Institute of Biotechnology, Shanghai, China) and collected prior to being washed twice with PBS. The cells (1×10⁶) were then stained with Annexin V-fluorescein isothiocyanate and propidium iodide labeling solution (BD Biosciences, Franklin Lakes, NJ, USA), and then analyzed using a flow cytometer (Beckman Coulter, Brea, CA, USA) and an Annexin V/Dead Cell Apoptosis kit for flow cytometry (V13241, Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

The migration and invasion of ccRCC cells were determined using a Transwell assay. Membranes (8 mm filter) were embedded with collagen (Millipore). UOK-117 or 786-O cells (2.5×10⁴) were plated in Transwell chambers on the membrane. These chambers were placed in a 24-well plate and incubated for 14 h at 37°C. The cells that migrated/invaded through the membrane were stained with the reagent using a Crystal Violet Staining Solution kit (Beyotime Institute of Biotechnology). The stained cells were photographed using an inverted microscope (AE2000; Motic Incoporation, Ltd., Causeway Bay, Hong Kong). After capturing images, the stain from the membrane was eluted in DMSO according to the manufacturer's instructions. The absorbance of the eluted stain was measured with a spectrophotometer at 590 nm. This measurement was used arbitrarily as an indicator of the number of cells that had migrated or invaded (19).

Equal numbers of cells were plated in each well of twelve-well culture plates. After the cells reached 70–80% confluence, a line was scratched in the middle of the well using a pipette tip to create a wound. Images of the denuded area were captured using an inverted microscope (Motic Incoporation) just after the denudation (time, 0 h). Following incubation for another 48 h, images were again captured. For quantitation, the area of the wound was measured and normalized to vehicle-treated controls. To investigate the effects of miR-21 on migration of UOK-117 and 786-O cells, the cells were transfected with pre-miR-21 or anti-miR-21 and their respective negative controls for 24 h, after which the wound was created and the images were captured at 0 h (20).

CSC sorting and culture were performed as previously described (21–23). Briefly, under standard conditions, ccRCC cells were grown in RPMI-1640 (glucose 4.5 g/l) supplemented with L-glutamine, sodium pyruvate, 10% FBS and penicillin/streptomycin, in a humidified atmosphere at 37°C under 5% CO₂. Under sphere culture conditions, parental cell lines or the floating spheres obtained from transfections were plated in serum-free defined media (SFDM), which consisted of low-glucose (1 g/l) RPMI-1640 supplemented with L-Glutamine, sodium pyruvate, penicillin/streptomycin (Wisent Bio Products, Saint-Jean-Baptiste, QC, Canada), 20 ng/ml basic fibroblast growth factor (FGF), 20 ng/ml EGF, and B27 (Invitrogen; Thermo Fisher Scientific, Inc.) using 24-well ultra-low attachment plates (Corning Inc., Tewksbury, MA, USA). Spheres were dissociated with trypsin every 5–7 days and split to a 1:3 ratio. For the sphere formation assay, 500 cells/well were seeded in an ultra-low attachment 96-well plate (Corning Inc.) in 100 µl SFDM/well. SFDM (25 µl/well) was added every day for 3 weeks. Wells were photographed at 4× magnification (Nikon Eclipse TS100; Nikon, Tokyo, Japan). Sphere number was determined by ImageJ program, version 1.37 (National Institutes of Health, Bethesda, MA, USA).

Data are presented as the mean ± standard deviation. Comparisons were made using one-way analysis of variance, and Student's t-test was used to analyze the differences between two experimental groups. Statistical analyses were performed using the SPSS version 17.0 software (SPSS, Inc., Chicago, IL, USA).. All experiments were repeated at least three times, and representative experiments are shown. P<0.05 was considered to indicate a statistically significant difference.

The expression of miR-21 mRNA was examined in UOK-117 and 786-O ccRCC cells and normal HK-2 cells by RT-qPCR. miR-21 mRNA expression in ccRCC cells was significantly higher compared with that in the normal HK-2 cells (Fig. 1A).

Furthermore, the relative expression of miR-21 was compared between cancer tissues and adjacent tissues, and it was demonstrated that cancer tissues had higher miR-21 expression (Fig. 1B).

To investigate the functional role of the upregulation of miR-21 in ccRCC cells, pre-miR-21 or anti-miR-21 was transfected into ccRCC cells, and their biological behavior was examined. Cell proliferation was analyzed using the MTT assay and flow cytometric analysis. Transfection of pre-miR-21 induced significantly increased growth rates in UOK-117 and 786-O ccRCC cells (Fig. 1C and D), and it was clearly shown that the two types of ccRCC cells displayed a decreased apoptotic rate compared with the controls (Fig. 2) (24).

Next, the role of miR-21 in regulating migration of ccRCC cells was determined using a Transwell chamber assay and wound scratch assay. Transfection with pre-miR-21 showed marked invasion, and anti-miR-21 blocked this invasion (Figs. 3 and 4). Quantification of these results showed significant changes in the ability to invade. Together these results demonstrated that miR-21 facilitates invasion of the two types of ccRCC cells (19).

Cells overexpressing miR-21 exhibited accelerated invasion, as indicated in wound healing and invasion chamber assays. Overall, these data support miR-21 as an essential target for mediating the growth and invasiveness of ccRCC cells in vitro.

UOK-117 and 786-O cells were transfected with pre-miR-21 or anti-miR-21. Western blotting was performed to determine EMT marker protein expression levels (fibroactin, N-cadherin, vinmentin and E-cadherin). The levels of all the proteins were upregulated in UOK-117 and 786-O cells, compared with controls, respectively (Fig. 5). These data suggested that miR-21 can promote EMT of UOK-117 and 786-O cells.

UOK-117 and 786-O cells were cultured in SFDM which was shown to support the formation of CSC spheres. Under these conditions, sphere formation was observed from the two ccRCC cell lines (Fig. 6A). Spheres could be propagated in three dimensional (3D) cultures in an ultra-low adherent dish for several passages. The ability of cell lines to form spheres in vitro depends on the presence of a self-renewing cell population. To assess the clonogenic potential of these sphere-forming cells, a single cell suspension prepared from UOK-117 and 786-O spheres and the parental cell lines was plated in SFDM. Large spheres were observed in 4–6 weeks. The sphere-derived cells typically formed >2 fold more spheres than the parental cell lines.

The role of miR-21 in cancer initiation and progression remains controversial. To determine the possible effect of miR-21 on ccRCC cancer sphere formation, ccRCC cell lines were transiently transfected with either pre-miR-21 or anti-miR-21. Transfection of UOK-117 and 786-O cells with pre-miR-21 led to rapid formation of 3D spheres (Fig. 6B), which were morphologically indistinguishable from the spheres obtained in SFDM (Fig. 6A). To confirm the ability of miR-21 to facilitate self-renewal, the clonogenic capacity of UOK-117 and 786-O cells was quantified upon miR-21 or treatment with transfection agent. miR-21 resulted in 1.9-fold increase in the number of colonies in the UOK-117 cell line and 1.5-fold increase in the number of colonies in the 786-O cell line compared with the transfection agent control (Fig. 6C and D).