Twenty-five pairs of human RCC and adjacent normal tissues were surgically collected from patients at Ningbo Urology and Nephrology Hospital from March, 2015 to December 2017. Among the 25 enrolled patients, 12 were male, 13 were female and the mean age was 62.4±5.5 years. Before surgery, none of the patients received any chemotherapy or radiotherapy. The clinicopathological features were recorded based on the American Joint Committee on Cancer (AJCC) standards (12). All patients provided written informed consent and the study was approved by the Ethics Committee of Ningbo Urology and Nephrology Hospital (Ningbo, China).

Cells (293) were purchased from the Shanghai Institute for Biological Sciences (Shanghai, China). Human RCC cells (786-O, ACHN and Caki-1) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). 786-O and Caki-1 cells were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). ACHN and 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.). Media were supplemented with 10% fetal bovine serum (FBS; Gibco Thermo Fisher Scientific, Inc.), 1% antibiotics (100 µl/ml penicillin and 100 mg/ml streptomycin sulfate; Gibco; Thermo Fisher Scientific, Inc.) and 1% glutamine (Gibco; Thermo Fisher Scientific, Inc.). Cells were cultured in a humidified incubator containing 5% CO₂ at a temperature of 37°C. The Cell Counting Kit-8 (CCK-8) assay was performed to assess cell viability. Briefly, cells (5,000 cells/well) were seeded in a 96-well plate. Twenty-four hours after transfection, 10 µl of CCK-8 solution (Beyotime Institute of Biotechnology, Shanghai, China) was added, and 1 h later, the optical density (OD) value of each well was measured with an ELISA microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA) at a wavelength of 595 nm. Wells without cells were used as blanks. The experiments were performed in triplicate and repeated at least three times.

Total RNA was extracted from the tissue samples and cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), and purified with the RNeasy Maxi kit (Qiagen, Inc., Santa Clarita, CA, USA) according to the manufacturer's guidelines. RNA concentrations were measured using a NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Inc.). Reverse transcription to prepare cDNA templates was performed using a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Then, qPCR was performed with a miScript SYBR-Green PCR kit (Qiagen) and the LightCycler 480 Real-Time PCR system (Roche Diagnostics, Basel, Switzerland). GAPDH and U6 were used as internal controls for VEGFA and miR-205-5p, respectively. The following thermocycling conditions were used: 95°C for 1 min, then 40 cycles of 95°C for 15 sec, 55°C for 30 sec and 70°C for 30 sec. The expression levels in tissues and cells were calculated using the 2^−ΔΔCq method (13).

Synthesized miR-205-5p mimics (5′-UCCUUCAUUCCACCGGAGUCUG-3′) or the negative control (miR-NC) (5′-UCACAACCUCCUAGAAAGAGUAGA-3′) were purchased from Suzhou GenePharma Co., Ltd. (Suzhou, China). The myr-Akt vector and empty vector were generous gifts from Dr Rui Yu (Ningbo University, Ningbo, China). Cells (2×10⁵) were transfected with 20 µM miRNA mimics or 2 µg vector plasmid. Twenty-four hours after transfection, the cells were collected and assayed. Transfection was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions.

Cells were seeded in a 6-well plate and allowed to grow to 90% confluence to assess migration in vitro. Twenty-four hours after transfection, an artificial wound was created with a 200-µl pipette tip in the center of the confluent cell monolayer. Then, the cells were cultured for another 24 h and the closure of the wound in each group was evaluated at the magnification of ×2,000 under an inverted microscope (Olympus Corp., Tokyo, Japan). For the invasion assay, 1×10⁵ cells were suspended in 200 µl of serum-free medium and plated in upper Transwell chambers (Costar; Corning Inc., Corning, NY, USA) coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). Six hundred microliters of serum-containing medium were added to the bottom well. After culture in a humidified atmosphere containing 5% CO₂ at 37°C for 24 h, the cells that had migrated and adhered to the lower surface of the membrane were fixed with 70% methanol and stained with 1% crystal violet for 15 min. Finally, the number of cells that migrated across the membrane were counted. Migration and invasion of cells was evaluated with an inverted Olympus phase-contrast microscope (×200 magnification) (Olympus Corp.). Each experiment was performed in triplicate.

The percentage of apoptotic cells was measured using Nucleosome ELISA kit (cat. no. 11544675001; Roche Diagnostics), according to the manufacturer's instructions. Briefly, cells were seeded into 24-well plates and transfected. Twenty-four hours later, the cells were collected and lysed. A biotin-labeled mouse antibody against histone 3 that specifically binds to the histone component of captured nucleosomes derived from apoptotic cells was used in this assay. The bound antibody was detected following an incubation with horseradish peroxidase (HRP)-conjugated streptavidin at room temperature for 1 h. HRP catalyzes the conversion of the colorless tetramethylbenzidine (TMB) to produce a blue color. The addition of stop solution turns the solution yellow, and the intensity was proportional to the number of nucleosomes in the sample. Each experiment was performed in triplicate.

Cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology). The protein concentration was determined by the Bradford assay kit according to the manufacturer's guidelines (Beyotime Institute of Biotechnology). Lysates were centrifuged at 12,000 × g 5 min at 4°C and stored at −70°C. Equal amounts (20 µg) of protein lysates were separated on 10% SDS-PAGE gels and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Minneapolis, MN, USA). PVDF membranes were blocked with 10% skim milk powder dissolved in TBST buffer at room temperature for 1 h. Then, PVDF membranes were stripped and washed with TBST, and then incubated with a primary antibody (dilution of 1:1,000) in TBST containing 5% bovine serum albumin (BSA) overnight at 4°C. Primary antibodies against the following proteins were used: MMP-7 (cat. no. ab207299), MMP-9 (cat. no. ab73734), Snail (cat. no. ab53519) (all from Abcam, Cambridge, MA, USA), N-cadherin (cat. no. 4061), caspase-3 (cat. no. 9662), Bcl-2 (cat. no. 3498), Bcl-xL (cat. no. 2762), p-PI3K (cat. no. 4228), t-PI3K (cat. no. 4225), p-Akt (cat. no. 4058), t-Akt (cat. no. 4691), p-mTOR (cat. no. 5536), t-mTOR (cat. no. 2983) (Cell Signaling Technology, Danvers, USA) and GAPDH (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Next, membranes were washed with TBST three times and incubated with an HRP-conjugated anti-rabbit (cat. no. RABHRP1) or anti-mouse secondary antibody (cat. no. RABHRP2) (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Signals were visualized using an ECL reagent (Pierce; Thermo Fisher Scientific, Inc.). All western blots were replicated in three times and the intensity of the western blot signals was analyzed using ImageJ software version 1.48 (NIH; National Institutes of Health, Bethesda, MD, USA).

The potential miR-205-5p binding sites in the VEGFA 3′-UTR were predicted using TargetScan 7.1 software (www.targetscan.org/). Sequences containing the wild-type (VEGFA-wt) or mutant (VEGFA-mut) seed region of VEGFA were synthesized and cloned into a luciferase reporter plasmid (pMIR-REPORT) (Promega Corporation, Madison, WI, USA). The VEGFA-wt or VEGFA-mut plasmids and miR-205-5p or miR-NC were co-transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Twenty-four hours after transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase assay (Promega Corporation) according to the manufacturer's instructions. All luciferase assays were performed in triplicate.

The animal study was approved by the Ethics Committee on Animal Research of Ningbo Urology and Nephrology Hospital (Ningbo, China). Male nude mice (BALB/c, 4–5 weeks old) were purchased from the Shanghai Laboratory Animal Center, Shanghai Institute for Life Sciences, Chinese Academy of Sciences (Shanghai, China). The total number of mice were 50 and the weight of the mice was 20±5 g. The mice were housed in a facility at 23–24°C, and the light-dark cycle was set at 12-h intervals. The miR-205-5p-overexpressing 786-O cells or miR-NC-overexpressing 786-O cells were prepared by transfecting the cells with a recombinant lentivirus carrying the miR-205-5p precursor sequence or a scrambled control, respectively (Shanghai GenePharma Co., Ltd., Shanghai, China). Cells were suspended in 100 µl of phosphate-buffered saline (PBS) and subcutaneously injected into the flank of a nude mouse. The width and length of the tumor nodules were measured every three days. The volume of subcutaneous tumors was calculated as tumor volume = length width × width/2. At 31 days post-injection, the mice were removed from their cages and gently restrained while resting on the benchtop. Cervical dislocation was performed manually and resulted in euthanasia within ~10 sec and the tumors were excised for use in subsequent experiments.

All statistical analyses were conducted using the SPSS 20.0 software (IBM Corp., Armonk, NY, USA). All measurement data are presented as means ± standard deviations (SDs). Univariate Cox proportional hazards regression analyses were performed to identify any significant variables predicting survival status. Multivariate Cox proportional hazard analyses were performed to assess the independent predictors of survival. One-way analysis of variance (ANOVA) with post hoc Tukey's test was chosen to compare data between multiple groups and paired Student's t-test was used to compare data between two groups. P<0.05 was considered indicative of a significant difference.

We examined the endogenous expression of miR-205-5p in RCC tissues (n=25) and adjacent normal kidney tissues (n=25) to determine whether miR-205-5p is dysregulated in RCC. Significantly lower expression of miR-205-5p was detected in RCC tumor tissues than that observed in the normal tissues (P<0.01; Fig. 1A). In addition, we measured the expression of miR-205-5p in the cell line 293 and RCC cell lines 786-O, Caki-1 and ACHN. Significantly lower expression of miR-205-5p was observed in RCC cell lines than in the 293 cells (Fig. 1B). Then, we divided the 25 samples into a high miR-205-5p expression group (n=13) and low miR-205-5p expression group (n=12) according the median expression to further evaluate the relationship between miR-205-5p expression and the clinicopathological features of RCC. As shown in Table I, a lower miR-205-5p level was associated with an advanced tumor stage, Fuhrman stage and more lymph node metastasis, but was not associated with age, sex or tumor size. In addition, the Kaplan-Meier analysis revealed that downregulation of miR-205-5p was associated with a poor prognosis, indicating that miR-205-5p may serve as a promising candidate prognostic biomarker for patients with RCC (mean follow-up time of 42 months) (Fig. 1C). A univariate analysis using the Cox proportional hazard regression model revealed statistically significant correlations between the overall survival of patients with miR-205-5p expression (P=0.017) and tumor stage (P=0.023) (Table II). A multivariate analysis including miR-205-5p expression and the tumor stage showed that miR-205-5p (P=0.024) and tumor stage (P=0.034) were independent prognostic factors for the overall survival of patients with RCC (Table II).

The 786-O and ACHN cells were transfected with the miR-205-5p mimic or miR-NC to investigate the function of miR-205-5p in RCC. We performed RT-qPCR to analyze the miR-205-5p expression in the transfected cells. The expression of miR-205-5p was significantly upregulated in both 786-O (P<0.001) and ACHN (P<0.001) cells at 24 h after transfection (Fig. 2A). The CCK-8 assay was employed to evaluate cell proliferation. Overexpression of miR-205-5p significantly suppressed the proliferation of both 786-O and ACHN cells compared with cells transfected with miR-NC (Fig. 2B). Thus, miR-205-5p suppressed the proliferation of RCC cells. Then, a scratch assay was performed to assess cell migration. Based on the results of the scratch assay, the migratory distance was markedly reduced in both cell lines transfected with the miR-205-5p mimic (Fig. 2C). Thus, miR-205-5p suppresses the migration of RCC cells.

Next, we assessed the effects of miR-205-5p on the invasion of RCC cells using Transwell assays. As shown in Fig. 3A, overexpression of miR-205-5p decreased the invasive ability of both 786-O and ACHN cell lines. Meanwhile, the levels of invasion and migration marker proteins (MMP-7 and MMP-9) were also obviously decreased in cells overexpressing miR-205-5p (Fig. 3B). Since epithelial-mesenchymal transition (EMT) is an indispensable process for tumor cell invasion and migration (14), we examined whether miR-205-5p affects EMT in RCC cells. As shown in Fig. 3B, overexpression of miR-205-5p significantly decreased the protein levels of N-cadherin, β-catenin and Snail. Based on these data, miR-205-5p may inhibit the migration of RCC cells by suppressing the EMT. We also analyzed the numbers of apoptotic cells and found that overexpression of miR-205-5p significantly increased the percentage of apoptotic 786-O and ACHN cells (Fig. 3C). Furthermore, the levels of apoptosis-related proteins were examined using western blotting. As indicated in Fig. 3D, caspase-3, Bcl-2 and Bcl-xL levels were reduced in cells overexpressing miR-205-5p. Therefore, miR-205-5p likely suppresses the proliferation of RCC cells by inducing apoptosis.

RCC is normally resistant to conventional cytotoxic chemotherapies. Therefore, we investigated the effect of miR-205-5p on the responses of RCC cells to various chemotherapeutic agents. First, we tested paclitaxel, an agent known to inhibit cell division and thereby induce cell death. Measurements of the half maximal inhibitory concentration (IC₅₀) showed an increased sensitivity of 786-O and ACHN cells to paclitaxel (Fig. 4A). Similar results were also obtained with 5-FU and oxaliplatin, agents that block DNA replication (Fig. 4B and C). We also tested sunitinib, a receptor tyrosine kinase (RTK) inhibitor used to treat patients with advanced ccRCC (15). Notably, overexpression of miR-205-5p also increased the sensitivity of 786-O and ACHN to sunitinib (Fig. 4D). Therefore, miR-205-5p is also involved in the response of RCC cells to chemotherapy.

The PI3K/Akt/mTOR signaling pathway exerts significant effects on the proliferation, invasion and apoptosis of RCC cells (16). We, therefore, investigated the effects of miR-205-5p on the PI3K/Akt signaling pathway. As indicated in Fig. 5A, overexpression of miR-205-5p markedly decreased p-PI3K, p-Akt and p-mTOR levels. Constitutively active Akt (myr-Akt) was overexpressed as described in a previous study to further elucidate the role of the PI3K/Akt pathway in the antitumor effects of miR-205-5p (17). Overexpression of myr-Akt successfully mimicked the activation of Akt (Fig. 5B). Myr-Akt also significantly suppressed miR-205-5p-induced apoptosis (Fig. 5C). Based on these data, the tumor suppressor function of miR-205-5p in RCC is likely and at least partially mediated by its repression of the PI3K/Akt/mTOR pathway.

According to bioinformatic analysis tools (TargetScan; www.targetscan.org and miRanda; www.microrna.org), we speculated that VEGFA is a candidate target of miR-205-5p. As shown in Fig. 6A, the RT-qPCR analysis revealed significantly decreased levels of the VEGFA mRNA in 786-O and ACHN cells transfected with miR-205-5p. Western blot analyses also showed decreased levels of the VEGFA protein in 786-O and ACHN cells transfected with the miR-205-5p mimic (Fig. 6B). We performed a Dual-Luciferase assay to further confirm that VEGFA is a target of miR-205-5p. The wild-type (WT) and mutant (Mut) VEGFA 3-untranslated region (3′-UTR) constructs were subcloned into the pMIR reporter plasmid. Overexpression of miR-205-5p decreased luciferase activity in both 786-O and ACHN cells transfected with the wild-type (WT) 3′-UTR of VEGFA, but not the mutant (Mut) 3′-UTR (Fig. 6D). Levels of the VEGFA mRNA in RCC and normal adjacent tissues were examined using qRT-PCR to elucidate the clinical relevance of miR-205-5p-mediated targeting of VEGFA in RCC. As shown in Fig. 6E, the VEGF mRNA was expressed at much higher levels in RCC tumor tissues than that noted in normal tissues. Furthermore, an inverse correlation was observed between the expression of miR-205-5p and VEGFA expression in RCC tissues (r=−0.179) (Fig. 6F). These results further confirmed that VEGFA expression is negatively regulated by miR-205-5p in RCC. In summary, VEGFA is a target of miR-205-5p in RCC.

We stably expressed miR-NC or miR-205-5p in 786-O cells and then subsequently implanted these cells into both posterior flanks of nude mice to investigate whether overexpression of miR-205-5p attenuates the progression of RCC in vivo. Tumor sizes were measured after 7 days. From the 22nd to the 31st day, the miR-NC group developed significantly larger tumors than the miR-205-5p group (Fig. 7A). Meanwhile, the final tumor weight of the miR-NC group was much greater than the miR-205-5p group (Fig. 7B). Consistent with the results from the in vitro studies, lower levels of the caspase-3, Bcl-2 and Bcl-xL proteins were detected in the tumor tissues from the miR-205-5p-expressing group than in the miR-NC-expressing group (Fig. 7C). Based on these data, miR-205-5p also inhibits tumor growth in vivo.