A total of 28 tumor samples and matched non-tumor kidney tissues were obtained from Peking University Shenzhen Hospital (Shenzhen, China) between January 2015 and January 2016. The normal kidney tissue was obtained by sampling 2 cm away from the tumor tissue. The specimens were frozen in liquid nitrogen (−195.8°C) until RNA extraction. The present study was approved by the local Ethics Committee of Peking University Shenzhen Hospital (Shenzhen, China), and written informed consent was obtained from all patients. The clinic pathological features of all patients are listed in Table I.

Total RNA was extracted from the RCC tissue and adjacent tissue using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and was purified using the RNeasy Maxi kit (Qiagen GmbH, Hilden, Germany) following the manufacturer's protocols. The concentration of the RNA was measured using a NanoDrop 2000/2000c spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA). The miScript Reverse Transcription kit (Qiagen GmbH) was used to prepare cDNA from 1 µg RNA. The reverse transcription process consisted of 37°C for 60 min, 95°C for 5 min and then storage at 4°C. To analyze the expression of miR-136-5p, qPCR was conducted using the resultant cDNA with the miScript SYBR^® Green PCR kit (Qiagen GmbH) following the manufacturer's protocol, on the Roche Light Cycler 480 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland). The thermocycler conditions were as follows: 95°C for 1 min, 40 cycles of 95°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec. The sequences of the primers for miR-136-5p and the internal control, U6, are presented in Table II. The 2^−ΔΔCq method was used to analyze the expression of miR-136-5p (14).

A normal human embryo kidney cell line (293T) and two RCC cell lines (786-O and ACHN) were obtained from the Guangdong and Shenzhen Key Laboratory of Male Reproductive Medicine and Genetics (Shenzhen, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin-streptomycin (Gibco; Thermo Fisher Scientific, Inc.) and 1% glutamine (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a 5% CO₂ humidified incubator. Transfection was performed with miR-136-5p mimics, miR-136-5p inhibitors, negative control (NC) miRNA and inhibitor negative control (inhibitor NC) miRNA (5 pmol; Shanghai GenePharma Co., Ltd., Shanghai, China) using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol when cells were at 70% confluence. The sequences of mimics, inhibitor, NC and inhibitor NC are presented in Table II. Following transfection, RT-qPCR was used to assess transfection efficiency and the expression of miR-136-5p.

Cells (5×10³) transfected with 5 pmol miR-136-5p mimics, inhibitors, NC or inhibitor NC were seeded in 96-well plates and then incubated at 37°C. After 0, 24, 48 and 72 h, 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology, Haimen, China) was added into each well of a 96-well plate for 30 min at 37°C, and then the absorbance values of the experimental wells were read at 490 nm.

Following transfection with 5 pmol miR-136-5p mimics, NC, miR-136-5p inhibitors or inhibitor NC, ~5×10³ cells were seeded into each well of a 96-well plate and incubated for 4 days. Following addition of 20 µl MTT (5 mg/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) to the plate, the cells were incubated for a further 4 h at 37°C. The medium was discarded, 100 µl DMSO was added into each well, and the plates were shaken on a reciprocating decolorization shaking table (TSB-108 Qilinbeier, Jiangsu, China; http://www.qilinbeier.cn/tsb-108.html) for 10 min in the dark. Then, the absorbance values were read at 595 nm using an ELISA microplate reader (680; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cells (5×10⁵) were seeded into each well of a 12-well plate, and incubated until they formed a monolayer of ~80% confluent cells. Cells were then transfected with 100 pmol chemically synthesized miRNA-136-5p inhibitors, mimics, NC or inhibitor NC, as aforementioned. A wound was created with a sterile 200 µl pipette tip, and floating cells were washed away using phosphate-buffered saline (PBS). Cell images were taken at 0 and 12 h after making the scratch using a digital camera system, and the temperature of incubation subsequent to making the scratch was 37°C.

Transwell chamber inserts (BD Biosciences, Franklin Lakes, NJ, USA) with or without Matrigel (for invasion assays) were used. Serum-free medium (200 µl DMEM) containing ~1×10⁴ transfected cells was seeded into upper chamber of the insert. Medium mixed with 10% FBS was added to the lower chamber of the inserts. 786-O cells and ACHN cells were incubated at 37°C for 36 h for the migration assay, and were incubated for 48 and 60 h, respectively, for the invasion assay. All non-migrated cells were removed by a cotton swab. The cells that had migrated or invaded to the other side of the membrane were stained with 0.1% crystal violet for 25 min at 25°C and counted for in three fields of view using a microscope (Leica DMIRB Inverted Fluorescence Microscope, Leica Microsystems GmbH, Wetzlar, Germany).

Cells were seeded in a 6-well plate (3×10⁵ cells/well) and maintained in a humidified incubator with 5% CO² at 37°C until they formed an ~80% confluent monolayer. Cells were then transfected with 200 pmol miR-136-5p mimics, inhibitors, NC or inhibitor NC. Following transfection for 48 h, cells were gathered and washed twice with cold PBS. Then, the cells were re-suspended in 100 µl 1X binding buffer (Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit, Invitrogen; Thermo Fisher Scientific, Inc.) to create a single-cell suspension. Cell suspension (50 µl), 5 µl Annexin V-fluorescein isothiocyanate and 5 µl propidium iodide (Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit) were mixed and then incubated in room temperature for 15 min in the dark Following the addition of 400 µl binding buffer, the samples were analyzed using a flow cytometer (EPICS, Xl-4; Beckman Coulter, Inc., Brea, CA, USA), and the software used for data analysis was FlowJo v 10 (TreeStar, Inc., Ashland, OR, USA).

Potential targets of miR-136-5p were predicted through the combination of four public algorithms, including miRWalk (15) (www.umm.uni-heidelberg.de/apps/zmf/mirwalk), miRanda (16) (www.microrna.org), PicTar (pictar.mdc-berlin.de) and TargetScan (17) (www.targetscan.org). All four algorithms accepted the predicted genes, which were selected based on gene function.

Each experiment was performed in triplicate and repeated at least three times. The data are presented as the mean ± standard deviation. MiR-136 expression levels between groups were analyzed using paired Student's t-tests, with the exception of relative expression of miR-136-5p in cells as presented in Fig. 1, which was analyzed using one-way analysis of variance followed by Dunnett's post hoc test. A paired Student's t-test was also used to analyze the results of assays to characterize cell phenotype. All statistical calculations were performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was performed to examine the expression levels of miR-136-5p in 28 paired RCC tissues and cell lines. As presented in Fig. 1A, the ratio of miR-136-5p expression [log₂Ratio (T/N)] was revealed to be downregulated in the RCC tissues from 22/28 patients. Mean expression levels are presented in Fig. 1B, and revealed that the expression of miR-136-5p in RCC tissues was significantly decreased compared with adjacent normal tissues (P<0.01). Furthermore, miR-136-5p expression levels were significantly decreased in ACHN (P<0.05) and 786-O (P<0.05) RCC cell lines compared with the 293T normal kidney human embryo kidney cell line, as presented in Fig. 1C.

qPCR was performed to assess the transfection efficiency of miR-136-5p mimics or NC and miR-136-5p inhibitors or inhibitor NC. Compared with the NC group, the expression levels of miR-136-5p were 145.00 times higher in ACHN cells (P<0.01) and 67.33 times higher in 786-O cells (P<0.001) transfected with miR-136-5p. Compared with the inhibitor NC group, miR-136-5p expression levels were 0.16 times that in ACHN cells (P<0.01) and 0.09 times that in 786-O cells (P<0.001) following transfection with miR-136-5p inhibitors (Fig. 1D).

The effect of miR-136-5pon proliferation was determined using a CCK-8 assay (Fig. 2). Following transfection with miR-136-5p mimics, the proliferation of ACHN cells was decreased by 13.97% (24 h; P<0.01), 28.15% (48 h; P<0.01) and 19.21% (72 h; P<0.01), while that of 786-O cells was decreased by 6.67% (24 h), 14.35% (48 h; P<0.05) and 17.62% (72 h; P<0.001), respectively, compared with the NC group (Fig. 2A and C). Furthermore, following transfection with miR-136-5p inhibitors, the proliferation of ACHN cells was increased by 10.15% (24 h; P<0.05), 28.30% (48 h; P<0.01) and 40.19% (72 h; P<0.01), while that of 786-O cells was increased by 8.29% (24 h; P<0.05), 11.97% (48 h; P<0.05) and 20.45% (72 h; P<0.001), respectively, compared with the inhibitor NC group (Fig. 2B and D).

The effect of miR-136-5p on cell viability was determined using an MTT assay. As presented in Fig. 2E, the viability of ACHN cells transfected with the miR-136-5p mimic was reduced by 12.89% (P<0.01) compared with the NC group, while the viability of cells transfected with the miR-136-5p inhibitor was increased by 16.39% (P<0.01) compared with the inhibitor NC group. Similarly, the viability of 786-O cells transfected with the miR-136-5p mimic was decreased by 16.71% (P<0.05) compared with the NC group, while the viability of cells transfected with the miR-136-5p inhibitor was increased by 13.15% (P<0.05) compared with the inhibitor NC group (Fig. 2F).

The effect of miR-136-5p on RCC cell mobility was determined using wound healing assays and Transwell assays (Figs. 3 and 4). Representative images of the wound healing assay are presented in Fig. 3A. The wound healing assay revealed that, compared with the NC group, the migration of the mimic group was reduced by 28.53% (P<0.05; Fig. 3C) in ACHN cells and by 27.99% (P<0.05; Fig. 3B) in 786-O cells 12 h following the initial scratch. In contrast, compared with the inhibitor NC group, the migration of the inhibitor group was increased by 73.14% (P<0.01; Fig. 3C) in ACHN cells and by 31.08% (P<0.05; Fig. 3B) in 786-O cells 12 h following the initial scratch.

As presented in Fig. 4C, the Transwell assay revealed that, following transfection with miR-136-5p mimics, ACHN cell migration was reduced by 60.34% compared with the NC group (P<0.01), while the cells transfected with miR-136-5p inhibitors was increased by 100.26% compared with the inhibitor NC group (P<0.01). The invasion of ACHN cells transfected with miR-136-5p mimics was reduced by 75.52% compared with the NC group (P<0.01), while the invasion of cells transfected with miR-136-5p inhibitors was increased by 109.52% compared with the inhibitor NC group (P<0.05; Fig. 4C). Similarly, the migration of 786-O cells transfected with miR-136-5p mimics was reduced by 49.02% compared with the NC group (P<0.01), while the migration of cells transfected with miR-136-5p inhibitor was increased by 84.09% compared with the inhibitor NC group (P<0.05; Fig. 4B). The invasion of 786-O cells transfected with miR-136-5p mimics was reduced by 78.41% compared with the NC group (P<0.01), while the cells transfected with miR-136-5p inhibitors was promoted by 60.63% compared with the inhibitor NC group (P<0.01; Fig. 4B).

Flow cytometry was used to assess the effect of miR-136-5p on apoptosis (Fig. 5A). The results revealed that the early apoptosis rate of 786-O cells transfected with miR-136-5p mimics and NC was 27.56±0.40 and 16.73±0.26%, respectively (P<0.01; Fig. 5B), while the early apoptosis rate of 786-O cells transfected with miR-136-5p inhibitors and inhibitor NC was 12.93±0.34 and 17.66±0.32%, respectively (P<0.05; Fig. 5B). Similarly, the early apoptosis rate of ACHN cells transfected with miR-136-5p mimics and NC was 31.4±0.78 and 24.26±0.66%, respectively (P<0.05; Fig. 5C), while the early apoptosis rate of ACHN cells transfected with miR-136-5p inhibitors and inhibitor NC was 15.03±0.93 and 24.33±0.72%, respectively (P<0.05; Fig. 5C).

Four algorithms were combined to predict the putative target genes of miR-136-5p. All four algorithms simultaneously predicted that dedicator of cytokinesis 5 (DOCK5) was a potential target. The complementary site for the seed sequences of miR-136-5p was 5′-AAUGGAGA-3′ in the DOCK5 3′-untranslated region.