RCC tissues (n=52) and corresponding non-cancerous renal tissues (n=52) from patients that were pathologically diagnosed with RCC and had received no therapy were collected from the Peking University Shenzhen Hospital between January 2016 and June 2017. (Guangdong, China). The corresponding non-cancerous renal tissues were collected >2 cm away from the visible tumor range. Written informed consent was obtained from every patient. All the patients were pathologically reviewed and diagnosed with RCC and had not received any chemoradiotherapy. The present study was approved by the Ethics Committees of Peking University Shenzhen Hospital. The collected tissues were checked and classified by hematoxylin and eosin staining. Subsequently, the samples were immersed in RNAlater reagent (Qiagen GmbH, Inc., Hilden, Germany) for ~0.5 h and stored at −80°C. The clinical and pathological information of the patients is presented in Table I.

The FFPE RCC samples obtained from the Department of Pathology of Peking University Shenzhen Hospital originated from patients with RCC who had undergone surgery at Peking University Shenzhen Hospital between January 2011 and January 2014. For fixation, all the tissue sample was fixated with 10% formalin concentration at 4°C for 48 h. For RNA extraction, ~10-µm-thick FFPE sections were used. The clinical and pathological characteristics of the patients, which are presented in Table II, were analyzed on the basis of the 2010 American Joint Committee on Cancer staging system (19). Total RNA of FFPE samples was extracted using an miRNeasy FFPE Kit (Qiagen GmbH, Inc.).

The cells used in the present study included human embryo kidney cells (293T; Cell Bank of Type Culture Collection of the Chinese Academy of Medical Sciences, Shanghai, China) and RCC cell lines (786-O, ACHN and Caki-1; American Type Culture Collection, Manassas, VA, USA). Cells were maintained at 37°C in an atmosphere containing 5% CO₂. The composition of the medium was as follows: 89% Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), 0.5% antibiotics (100 µl/ml penicillin and 100 mg/ml streptomycin sulfates) and 0.5% glutamine. A total of 100 pmol miR-572 mimic, inhibitor, negative control (NC) and inhibitor negative control (all Shanghai GenePharma Co., Ltd., Shanghai, China) were transfected into RCC cells for 5 h using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Subsequent experiments were performed 24–48 h following transfection. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to evaluate the transfected effect. The sequences were presented in Table III.

Total RNA of tissues and cell lines (786-O, ACHN, Caki-1 and 293T) were extracted and purified with the using of TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the RNeasy Maxi Kit (Qiagen GmbH, Inc.), respectively, according to the manufacturer's protocol. For improved experimental results, the extractive RNA samples were measured using a NanoDrop 2000c (Thermo Fisher Scientific, Inc.) to ensure the concentration and the optical density ratio (260/280) of 1.8–2.0 were taken into account. Following this, the eligible RNAs were synthesized to cDNA with the miScript II RT Kit (Qiagen GmbH). qPCR reactions were performed to ascertain the expression levels of miR-572 using the Roche LightCycler 480 Real-Time PCR System with a miScript SYBR-Green PCR Kit (Qiagen GmbH) under the following conditions: 95°C for 1 min, followed by 40 cycles at 95°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec. RNU6B (U6) was used as internal reference for normalization. The primers used in the qPCR are indicated in Table III. Relative expression levels were calculated using the 2^−ΔΔCq method (20).

Assessment of the cell migratory aptitude was performed using the wound healing assay. In the assay, a 12-well plate was inoculated with 786-O and ACHN cells (~3×10⁵ cells/each well) transfected with miR-572 mimic, inhibitor, NC or inhibitor NC (Shanghai GenePharma, Co., Ltd.). At 6 h post-transfection, a scratch was introduced using a sterile 200-µl pipette tip. Subsequently, the floating cells were washed away using phosphate-buffered saline (PBS; Gibco; Thermo Fisher Scientific, Inc.). At 0, 12 and 24 h, A Leica DMIRB inverted fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany) with a digital camera system (Olympus Corporation, Tokyo, Japan) was used to capture images of the scratches.

Assessment of the proliferation capacity of 786-O and ACHN cells was performed using the CCK-8 assay. A total of 5 pmol of miR-572 mimics, inhibitors, NC or inhibitor NC was transfected into the cells that had seeded for 24 h in 96-well plats with 4×10³ cells/well. Following gene transfection, 10 µl CCK-8 (Everbright lnc., Sayreville, NJ, USA) was added to each well and incubated for 0, 24, 48 and 72 h. The optical density value of each well was calculated 1 h later with an ELISA microplate reader (model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a wavelength of 490 nm.

The Transwell assay was used to assess the migratory and invasive aptitude of ACNH and 786-O cells. According to the manufacturer's recommendation, Transwell chamber inserts (BD Biosciences, New York, NJ, USA) with Matrigel were used to determine cell migration, whereas the inserts without Matrigel were used to assay cell invasion. A total of 200 µl serum-free DMEM containing 2×10⁴ transfected cells was added into the upper chamber of the inserts, whereas 500 µl DMEM with 10% FBS was added into the lower chamber of the inserts. Following 48 h of incubation, the cells migrated or invaded to the lower chamber of the inserts were stained with crystal violet at room temperature for 25 min, washed with PBS three times and counted using a microscope under the white light at a magnification of ×100 (Leica Microsystems GmbH), successively.

Assessment of cell apoptosis rates depended on flow cytometry assay. The cells plated in 6-well plates with ~3×10⁵ cells/plate were transfected with 200 pmol of miR-572 mimics, miR-572 inhibitors, NC inhibitor or NC according to the manufacturer's instructions. Following incubated for 48 h, all cells were harvested, washed by cold PBS twice and re-suspended in 100 µl 1X binding buffer. Subsequently, the re-suspended cells were mixed with 5 µl propidium iodide (Invitrogen; Thermo Fisher Scientific, Inc.) and 5 µl Annexin V-fluorescein isothiocyanate (Invitrogen; Thermo Fisher Scientific, Inc.), and cultured in darkness at room temperature for 15 min. Each tube was treated with 400 µl binding buffer. The apoptotic rates were measured using a flow cytometer (EPICS, Xl-4; Beckman Coulter, Inc., Brea, CA, USA). FlowJo software (version, X; FlowJo LLC, Ashland, OR, USA) was used for data analysis.

All assays were repeated three times. All statistical analysis was executed with the use of SPSS 19.0 (IBM Corp., Armonk, NY, USA). Paired Student's t-test was applied to assess the significance of the differences between two groups of data in matched tumor and non-cancerous tissues. The significance between cell lines was analyzing using one-way analysis of variance and Dunnett's post hoc test. Student's t-test was used the analysis of assays for characterizing phenotypes of cells. Analysis of the correlation between miR-572 expression and clinicopathological variables was performed using Fisher's exact test or Pearson's χ² test. The univariate and multivariate levels between miR-572 expression and clinicopathological variables or survival were determined using Cox proportional hazard regression analysis. The survival curves were plotted with Kaplan-Meier curves. The log-rank test was applied to evaluate the differences between these curves. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was performed with 52 pairs of RCC tissues and corresponding non-cancerous renal tissues with the purpose of exploring the clinical association of miR-572 in RCC. As indicated in Fig. 1A, the relative expression levels of miR-572 in 52 paired tissues [log2(T/N)]) were demonstrated and a total of 39 RCC tissues exhibited upregulation of miR-572. Fig. 1B indicated the mean expression of miR-572 and the results suggested that miR-572 was significantly upregulated in RCC tissues (5.515±0.263) compared with corresponding non-cancerous renal tissues (1.000±0.179; P<0.001). The relative expression levels of miR-572 in RCC cell lines (786-O, Caki-1 and ACHN) and 293T cell lines were also assessed (Fig. 1C). The results indicated that the expression levels of miR-572 were significantly increased in RCC cell lines (786-O, 8.084±0.618, P<0.01; ACHN, 9.372±1.371, P<0.01; and Caki-1, 4.643±0.793, P<0.05) compared with 293T cells (1.000±0.265). These results indicated that miR-572 may be up regulated in RCC tissues and possibly functions as an oncogene in RCC.

As indicated in Fig. 1C, the expression of miR-572 in ACHN and 780-O was most different from 293T. Subsequently, ACHN and 780O cells were selected for analysis in further functional assays. Validation of the transfection efficiency of miR-572 mimic or inhibitor was assessed using RT-qPCR. The outcomes revealed that the expression levels of miR-572 in cells transfected with miR-572 mimic were 79.893 times higher (786-O cells, P<0.01) and 67.649 times higher (ACHN cells, P<0.01) compared with NC. The expression levels of miR-572 in cells transfected with miR-572 inhibitor exhibited the opposite effect and were 0.119 times higher (786-O cells, P<0.05) and 0.418 times higher (ACHN cells, P<0.05) compared with NC inhibitor (Fig. 1D).

The role of miR-572 in cell proliferation was investigated using the CCK-8 assay. The outcomes indicated that the proliferation of 786-O significantly increased by 33.74% (P<0.01), 37.58% (P<0.01) and 24.72% (P<0.001) following transfection with miR-572 mimic compared with NC. However, proliferation was significantly decreased by 25.18% (P<0.05), 33.90% (P<0.001) and 10.01% (P<0.001) following transfection with miR-572 inhibitor compared with NC inhibitor at 24, 48 and 72 h (Fig. 2A and B). Similar outcomes were obtained for ACHN cells. Notably, the proliferation was significantly increased by 30.99% (P<0.01), 32.39% (P<0.001), 16.27% (P<0.001) but significantly decreased by 79.54% (P<0.01), 3.37% (P<0.01), 57.70% (P<0.001) at 24, 48 and 72 h following transfection with miR-572 mimic or inhibitor compared with the respective NC (Fig. 2C and D). These outcomes demonstrated that miR-572 may promote cell proliferation.

Assessment of the role of miR-572 of cell mobility was performed using wound healing assays and Transwell assays (Figs. 3 and 4). In the wound healing assay (Fig. 3A), 786-O and ACHN cells transfected with miR-572 mimic exhibited significantly increased migration compared with NC (Fig. 3B and C; 786-O, 1.862±0.096 vs. 1.000±0.092, P<0.01; ACHN, 1.589±0.038 vs. 1.000±0.038, P<0.01), whereas transfection with miR-572 inhibitor had an opposite result when compared with NC (Fig. 3B and C; 786-O, 0.756±0.052 vs. 1.000±0.079, P<0.05; ACHN, 0.609±0.034 vs. 1.000±0.051, P<0.05). In the Transwell assay (Fig. 4A), the number of relative migratory 786-O and ACHN cells per field was significantly increased following treatment with miR-572 mimic (Fig. 4C; 786-O, 1.717±0.053 vs. 1.000±0.046, P<0.01; Fig. 4E; ACHN, 1.642±0.064 vs. 1.000±0.060, P<0.001). However, the number of migratory cells was significantly decreased following treatment with miR-572 inhibitor (Fig. 4C and E, respectively; 786-O, 0.656±0.060 vs. 1.000±0.079, P<0.05; and ACHN, 0.609±0.070 vs. 1.000±0.062, P<0.05). Notably, elevated levels of miR-572 significantly promoted cell invasion compared with the NC (Fig. 4B; 786-O, 1.626±0.048 vs. 1.000±0.133, P<0.05; Fig. 4D; ACHN, 1.667±0.043 vs. 1.000±0.074, P<0.01), whereas these effects were significantly reversed by miR-572 inhibitor when compared with NC inhibitor (Fig. 4B, 786-O, 0.680±0.065 vs. 1.000±0.043, P<0.05; Fig. 4D; ACHN, 0.635±0.091 vs. 1.000±0.047, P<0.01). These outcomes demonstrated that miR-572 may promote cell mobility.

Assessment of cell apoptosis was performed using flow cytometry. Elevated levels of miR-572 significantly inhibited cell early apoptosis compared with NC (786-O, 4.73±0.635% vs. 10.17±0.404%, P<0.001; ACHN, 4.52±0.800% vs. 10.22±0.595%, P<0.01), whereas these effects could be significantly reversed by miR-572 inhibitor (786-O, 18.87±0.862% vs. 10.26±0.530%, P<0.001; ACHN, 10.30±0.367% vs. 18.96±0.306%, P<0.001; Fig. 5). These outcomes demonstrated that miR-572 may inhibit early apoptosis in cells.

The association between the expression levels of miR-572 and clinicopathological variables or overall survival was analyzed with the FFPE renal cancer samples. As presented in Table II, no significant difference was observed between miR-572 expression level and sex, age, tumor size and tumor stage. However, univariate analysis (Fig. 6A) suggested that the expression level of miR-572 was inversely correlated with overall survival (HR=0.195, 95% CI=0.042–0.903, P=0.037; Table IV). Similar outcomes were indicated in the survival curves of Cox proportional hazard regression analysis (Fig. 6B), which suggested that the patients with low miR-572 expression levels had a significantly increased overall survival after adjusting for sex, age, tumor size and tumor stage in the multivariate analysis (HR=0.174, 95% CI=0.034–0.878, P=0.034; Table IV). Furthermore, data of the Kaplan-Meier survival curves demonstrated that the patients with low expression of miR-572 had a significantly longer overall survival (P=0.019, Fig. 6C). These outcomes demonstrated that miR-572 may serve as a potential independent prognostic marker for RCC.