A total of 32 RCC tissues and paired normal tissues were collected from the Third Affiliated Hospital of Soochow University (Changzhou, China). Informed consent was obtained from all patients. The present study was approved by the Institutional Review Board of the Third Affiliated Hospital of Soochow University. All samples were frozen in liquid nitrogen immediately after surgery. The clinical information and pathological characteristics of the 32 patients with RCC are presented in Table I.

The 786-O and A498 human RCC cell lines, and human embryonic kidney (HEK) 293 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), 1% 100 U/ml penicillin and 1% 100 mg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a humidified incubator containing 5% CO₂.

Human CASC2 gene (NR_026939) was cloned into a pcDNA3.1(+) vector (Thermo Fisher Scientific, Inc.). CASC2 was amplified by polymerase chain reaction (PCR), then the PCR products were double-digested by restriction endonucleases of PmeI and NotI (Takara Bio, Inc., Otsu, Japan). Subsequently, the digested products were sub-cloned into the PmeI and NotI sites of the psiCHECK-2 luciferase vector. An empty vector served as a negative control (NC). The miR-21 mimics, 5′-UAGCUUAUCAGACUGAUGUUGA-3′, and NC mimics, 5′-CAGUACUUUUGUGUAGUACAA-3′ were obtained from Shanghai GenePharma Co., Ltd. (Shanghai, China). pcDNA3.1(+)-CASC2 over-expression vector (2 µg) or 2 µg pcDNA3.1(+) empty vector was transfected into 786-O or A498 cells, and 200 pmol miR-21 mimics or NC mimics were transfected into 786-O or A498 cells. The 786-O and A498 cells were transfected using Opti-MEM and Lipofectamine 2000 reagents (Invitrogen; Thermo Fisher Scientific, Inc.) once the cells reached 50–70% confluence, according to the manufacturer's protocols.

Total RNA of each sample (tissues and cells) was extracted with TRIzol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Synthesis of cDNA with reverse transcriptase was performed with the PrimeScript RT Enzyme Mix I kit (Takara Bio, Inc.). The reaction mixtures were incubated at 37°C for 60 min, 95°C for 5 min and then held at 4°C. MiScript SYBR Green PCR kit (Qiagen, Inc., Valencia, CA, USA) was used to conduct a qPCR analysis. The sequences of the primers (Invitrogen; Thermo Fisher Scientific, Inc.) used were as follows: miR-21, forward 5′-TTGACTGTTGAATCTCATGGCAA-3′, reverse primers were provided by the miScript SYBR Green PCR kit; CASC2, forward 5′-TACAGGACAGTCAGTGGTGGTA-3′, reverse 5′-ACATCTAGCTTAGGAATGTGGC-3′; and human GAPDH (which served as an internal control), forward 5′-TCAAGAAGGTGGTGAAGCA-3′ and reverse 5′-AGGTGGAGGAGTGGGTGT-3′. The qPCR reaction was set up in a total volume of 20 µl, consisting of 10 µl MiScript SYBR Green PCR Mix, 2 µl primers (forward and reverse), 1 µl cDNA template and 7 µl RNase-free water. The reactions were performed as follows: 95°C for 15 min, followed by 40 cycles at 95°C for 10 sec, 60°C for 30 sec and 72°C for 20 sec, using an Applied Biosystems 7900 HT PRISM Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR results were calculated using the ΔΔCq method (11).

Proliferation of 786-O and A498 cells was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). The cells (~2×10⁴) were seeded into a 96-well culture plate 24 h prior to transfection. After 0, 24, 48 or 72 h of transfection, 20 µl MTT (5 mg/ml) was added to each well, and the plates were incubated for 3 h at 37°C. Subsequently, the precipitate was solubilized with 150 µl dimethyl sulfoxide (Sigma-Aldrich), and absorbance was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay microplate reader (model 3550; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Experiments were repeated at least three times.

786-O and A498 cells were transfected with pcDNA3.1(+)-CASC2 vector or pcDNA3.1(+) empty vector, and miR-21 mimics or NC mimics. A total of 6 h post-transfection, a cell-scratch spatula was used to make a scratch in the cell monolayer. The cells were then washed three times with phosphate-buffered saline and were incubated at 37°C for 24 h. Images of the scratches were captured using a digital camera system (Leica DC 350FX; Leica Microsystems, Wetzlar, Germany) after the cells had been incubated for 0 and 24 h. The software program MIAS-2000 (Leica Microsystems) was used to determine the distance of migration. The experiments were performed in triplicate, repeated at least three times, and were analyzed in a double-blind fashion by at least two observers.

The potential miR-21 binding sites of CASC2 predicted by computer-aided algorithms were obtained from miRanda (http://www.microrna.org) and miRcode (http://www.mircode.org).

The RNA sequence of wild-type CASC2 (CASC2-WT) was amplified by PCR, and was inserted between the PmeI and NotI restriction sites in the 3′-UTR of the hRluc gene in the psiCHECK-2 luciferase vector (Promega Corporation, Madison, WI, USA). The primer sequences for CASC2 (Invitrogen; Thermo Fisher Scientific, Inc.) were as follows: Forward 5′-AGCGGGCTGCAGGGCTGCGGGCGCT-3′ and reverse 5′-TTGATTTAAAGTAATTAGCACATTC-3′. CASC2 RNA sequence with mutations in the putative binding site (CASC2-MUT) was also inserted into the hRluc gene in the psiCHECK-2 luciferase vector. CASC2-MUT was chemically synthesized by Shanghai GenePharma Co., Ltd. All recombinant plasmids were verified by DNA sequencing analysis.

For the dual-luciferase reporter assay, 786-O cells were seeded into 24-well plates, and were co-transfected with 0.8 µg CASC2-WT or 0.8 µg CASC2-MUT plasmids and 80 pmol miR-21 mimics or 80 pmol NC mimics using Lipofectamine 2000. A total of 24 h post-transfection, firefly and Renilla luciferase activities were detected using the GloMax^®-Multi Jr Single Tube Multimode Reader (Promega Corporation), according to the Dual-Luciferase Reporter Assay system (Promega Corporation). All experiments were performed in triplicate and repeated at least three times.

Experimental data are presented as the mean ± standard deviation, and were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). Data were analyzed using Student's t-test or one-way analysis of variance and the Student-Newman-Keuls method. P<0.05 was considered to indicate a statistically significant difference.

The expression levels of CASC2 were detected in 32 RCC specimens using qPCR. The expression levels of CASC2 were significantly downregulated in the 32 RCC tissues compared with in the adjacent normal tissues (Fig. 1A; P<0.05). The expression levels of CASC2 were also lower in the 786-O and A498 cell lines compared with in the human embryonic kidney HEK 293 cells (Fig. 1B; P=0.017 and P=0.033, respectively). These data suggest that CASC2 may function as a tumor suppressor gene in RCC development.

To explore the function of CASC2 in RCC cell proliferation and migration, MTT and wound scratch assays were performed in 786-O and A498 cell lines. Post-transfection with the pcDNA3.1(+)-CASC2 overexpression vector, the optical density (OD) of 786-O cells revealed that relative cell proliferation was significantly decreased by 8.21% at 24 h, 14.22% at 48 h and 21.1% at 72 h (P=0.001); the OD of A498 cells also demonstrated that proliferation was decreased by 6.36% at 24 h, 12.46% at 48 h and 16.32% at 72 h (P=0.005; Fig. 2A). Images of the wound scratches were captured at 0 and 24 h post-transfection; overexpression of CASC2 markedly inhibited the migration of 786-O and A498 cells. The distance of migration is presented in Fig. 2B. The relative rates of migration were 32% in 786-O cells and 47% in A498 cells (P=0.007 and P=0.009, respectively). These results indicate that CASC2 inhibits the proliferation and migration of RCC cells.

Previous studies have reported that lncRNAs may act as competing endogenous RNAs or as molecular sponges that modulate the concentration and biological function of miRNAs (12,13). Bioinformatics analysis was performed in order to identify the potential targeted miRNA of CASC2. The results revealed that miR-21 binding sites were present in CASC2 (Fig. 3A). To further confirm whether CASC2 was a direct target of miR-21, a dual-luciferase reporter assay was conducted in 786-O cells. Consistent with expectations, the relative luciferase activity was significantly inhibited by miR-21 in the CASC2-WT vector-transfected cells (P=0.008), whereas miR-21 exhibited no inhibitory effect in cells transfected with the CASC2-MUT vector (Fig. 3B). Furthermore, as shown in Fig. 3C, the RNA expression levels of CASC2 were significantly downregulated in the cells transfected with miR-21 mimics compared with in the NC mimics group (P<0.001 and P=0.005 in 786-O and A498 cells, respectively). These results suggest that CASC2 may be directly targeted by miR-21 in RCC.

CASC2 was identified as a direct target of miR-21 in RCC, as demonstrated by dual-luciferase reporter assays. The role of miR-21 in CASC2-induced inhibition on RCC cells remains unknown. As presented in Fig. 4A, the growth of 786-O and A498 cells was significantly increased (P=0.017 and P=0.011, respectively) following transfection with the pcDNA3.1(+)-CASC2 vector + miR-21 mimics as compared with the pcDNA3.1(+)-CASC2 vector. Additionally, over-expression of miR-21 and CASC2 could promote 786-O (P=0.042) and A498 (P=0.025) cell migration when compared with over-expression of CASC2 (Fig. 4B). These results strongly indicate that over-expression of miR-21 in RCC cells partially reverses the inhibitory effects of CASC2 on cell proliferation and migration.