Twenty-one paired ccRCC tissues and normal adjacent tissues (NATs) were obtained from patients (17 males, 4 females; age range, 42–75 years old; mean age, 59 years old) who underwent nephrectomy in Yidu Central Hospital of Weifang between August 2014 and March 2016. None of these patients were treated with chemotherapy or radiotherapy prior to nephrectomy. All of the tissue specimens were immediately frozen and stored in liquid nitrogen until further RNA isolation. This study was approved by the Ethics Committee of Yidu Central Hospital of Weifang, and written informed consent was collected from all patients before they participated in this research.

Two human ccRCC cell lines, A498 (21–23) and 786-O and one normal renal cell line (HK-2) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). All ccRCC cell lines were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (all from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). HK-2 cells were grown at keratinocyte-SFM (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with bovine pituitary extract and human recombinant epidermal growth factor (all from Gibco; Thermo Fisher Scientific, Inc.). All these cell lines were maintained at 37°C in a humidified incubator containing 5% carbon dioxide (CO₂).

miR-599 mimics, negative control miRNA mimics (miR-NC), small interfering RNA (siRNA) against the expression of HMGA2 (si-HMGA2) and negative control siRNA (si-NC) were designed and synthesised by Shanghai GenePharma Co., Ltd. (Shanghai, China). HMGA2 overexpression plasmid (pCMV-HMGA2) and empty pCMV plasmid were acquired from OriGene Technologies, Inc., (Beijing, China). Cells were inoculated into six-well plates and transfected with miRNA mimics, siRNA or plasmid by using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were performed to evaluate the transfection efficacy.

Total RNA was isolated from tissue samples or cells by using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. The concentration and purity of total RNA were determined using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Inc.). The cDNA of miR-599 was synthesised by using a TaqMan MicroRNA reverse transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Quantitative PCR (qPCR) was conducted to detect miR-599 expression by using a TaqMan MicroRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) with U6 snRNA as an internal control. To quantify the mRNA expression of HMGA2, we conducted reverse transcription with a PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan) followed by qPCR with a SYBR Premix Ex Taq™ kit (Takara Bio, Inc.). GAPDH was employed as an internal control for the mRNA level of HMGA2. Relative gene expression was analysed through the 2^−∆∆Ct method (24).

CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was applied to evaluate cell proliferation of ccRCC. Transfected cells were harvested at 24 h post-transfection. In each well of a 96-well plate, 3,000 transfected cells were plated and incubated at 37°C in a humidified incubator with 5% CO₂ for 0, 24, 48 and 72 h. At every time point, CCK-8 assay was performed in accordance with the manufacturer's protocol. Briefly, 10 µl CCK-8 reagent was added into each well, and then incubated at 37°C for another 2 h. The absorbance value of each well was detected at a wavelength of 450 nm with the ELISA microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cellular invasion ability was examined by using a Boyden chamber containing 24-well Transwell plates (Corning Costar, Corning, NY, USA) with 8 µm pore membranes. After transfection for 48 h, cells were collected, suspended in FBS-free DMEM and seeded in the upper chamber of the insert, which was pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). At 24 h post-incubation, the remaining cells at the upper side of the 8 µm pore membranes were wiped away with cotton swabs. The invasive cells that had invaded the bottom of the inserts were fixed in 4% cold paraformaldehyde and stained with 0.5% crystal violet. The stained cells were photographed and counted under an inverted microscope (Olympus Corporation, Tokyo, Japan) at ×200 magnification.

TargetScan (http://www.targetscan.org/) and microRNA (www.microrna.org) algorithms were used to predict the putative targets of miR-599. HMGA2 was predicted as a potential target of miR-599. Luciferase reporter assay was performed to further determine whether miR-599 could directly bind to the 3′-UTR of HMGA2. A total of 6×10⁴ cells were seeded in triplicates in 24-well plates. Luciferase reporter plasmids, namely, pGL3-HMGA2-3′-UTR wild-type (Wt) and pGL3-HMGA2-3′-UTR mutant (Mut), were designed and synthesised by Shanghai GenePharma Co., Ltd. When the cell density reached 70–80% confluence, miR-599 mimics or miR-NC was transfected into cells with pGL3-HMGA2-3′-UTR Wt or pGL3-HMGA2-3′-UTR Mut by using Lipofectamine 2000. After 48 h of incubation, a dual luciferase reporter assay system (Promega Corp., Madison, Wisconsin, USA) was adopted to measure the luciferase activity in accordance with the manufacturer's procedure. The firefly luciferase activity was normalised to Renilla luciferase activity.

Total protein was extracted from tissue samples or cells in an ice bath by using RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The protein concentration was detected by using a BCA protein assay kit (Bio-Rad Laboratories, Inc.). Equal amounts of proteins were subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA), which was then blocked with 5% skimmed milk dissolved in TBS containing 0.1% Tween-20 (TBST). Afterwards, the membranes were incubated with primary antibodies against HMGA2 (1:1,000 dilution, ab184616; Abcam, Cambridge, UK) or GAPDH (1:1,000 dilution, sc-47724; Santa Cruz Biotechnology, Inc.) overnight at 4°C, rinsed with TBST, further incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution, sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h and washed with TBST. The protein signals were then visualised with an ECL detection kit (GE Healthcare Life Sciences, Chalfont, UK) and analysed with Image J 1.41 (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as an internal control.

Data were expressed as mean ± standard deviation and analysed using SPSS v21.0 (SPSS Inc., Chicago, IL, USA). Each assay was repeated at least thrice. Differences between two groups were compared using t test, whereas differences between multiple groups were compared through one-way ANOVA. Student-Newman-Keuls test was used as a post hoc test. P<0.05 was considered to indicate a statistically significant difference.

miR-599 expression levels were detected in 21 paired ccRCC tissues and NATs. The results of RT-qPCR revealed that miR-599 was underexpressed in ccRCC tissues compared with that in NATs (P<0.05; Fig. 1A). The expression level of miR-599 was further examined in two ccRCC cell lines (A498 and 786-O) and one normal renal cell line (HK-2) by conducting RT-qPCR. As shown in Fig. 1B, miR-599 expression level decreased in the A498 and 786-O cell lines compared with HK-2 (P<0.05). These results suggest that the downregulation of miR-599 may be correlated with ccRCC progression.

To elucidate the effects of miR-599 in ccRCC development, we overexpressed miR-599 in A498 and 786-O cells. RT-qPCR analysis confirmed that miR-599 was markedly upregulated in A498 and 786-O cells transfected with miR-599 mimics (P<0.05; Fig. 2A). Then, CCK-8 assays were performed to determine the effects of miR-599 overexpression on the proliferation of A498 and 786-O cells. Ectopic expression of miR-599 significantly restricted the proliferation of A498 and 786-O cells compared with that in the cells transfected with miR-NC (P<0.05; Fig. 2B). Additionally, Transwell invasion assays were conducted to investigate the invasion abilities of A498 and 786-O cells transfected with miR-599 mimics or miR-NC. Upregulation of miR-599 resulted in the reduced invasion capabilities of A498 and 786-O cells (P<0.05; Fig. 2C). These data suggest that miR-599 may play tumour suppressive roles in ccRCC.

Bioinformatics analysis was performed to predict the putative targets of miR-599 and to investigate the molecular mechanism by which miR-599 affected the aggressive phenotype of ccRCC cells. Among the candidates, HMGA2 was selected for further confirmation (Fig. 3A) because it is aberrantly highly expressed in ccRCC and significantly associated with ccRCC progression (25–28). To identify whether HMGA2 is a direct target of miR-599, we subjected A498 and 786-O cells cotransfected with miR-599 mimics or miR-NC and pGL3-HMGA2-3′-UTR Wt or pGL3-HMGA2-3′-UTR Mut to luciferase reporter assays. As shown in Fig. 3B, enforced expression of miR-599 significantly decreased the luciferase activities of the wild-type 3′-UTR reporter plasmid in A498 and 786-O cells (P<0.05). However, this suppressive effect was abrogated in the mutant 3′-UTR reporter plasmid, in which the binding sequences mutated. RT-qPCR and western blot analysis were performed to detect the mRNA and protein expression of HMGA2 in A498 and 786-O cells transfected with miR-599 mimics or miR-NC and to evaluate the association between miR-599 and HMGA2 in ccRCC. The results indicated that restored miR-599 expression in the two ccRCC cell lines resulted in significantly reduced HMGA2 expression at both mRNA (Fig. 3C, P<0.05) and protein (P<0.05; Fig. 3D) levels. Therefore, HMGA2 is a direct target gene of miR-599 in ccRCC.

HMGA2 was validated as a direct target of miR-599 in ccRCC. As such, we hypothesised that the tumour suppressive roles of miR-599 in ccRCC might be exhibited by HMGA2 knockdown. To confirm this hypothesis, si-HMGA2 was transfected into A498 and 786-O cells to knock down endogenous HMGA2 expression. Western blot analysis revealed that HMGA2 was obviously downregulated in A498 and 786-O cells following transfection with si-HMGA2 (P<0.05; Fig. 4A). Subsequent CCK-8 and Transwell invasion assays demonstrated that the inhibition of HMGA2 prohibited the proliferation (P<0.05; Fig. 4B) and invasion (P<0.05; Fig. 4C) of A498 and 786-O cells. This phenomenon resembled the inhibitory effects of miR-599 overexpression on ccRCC cells, further suggesting that HMGA2 is a direct downstream target of miR-599 in ccRCC.

A rescue experiment was performed to evaluate whether HMGA2 is responsible for the inhibitory effects of miR-599 on ccRCC cells. A498 and 786-O cells were cotransfected with miR-599 mimics and pCMV-HMGA2 or empty pCMV plasmid. Western blot analysis was conducted to examine the transfection efficiency, and the results confirmed that the downregulation of HMGA2 caused by miR-599 overexpression was recovered after the A498 and 786-O cells were cotransfected with pCMV-HMGA2 (P<0.05; Fig. 5A). In addition, CCK-8 and Transwell invasion assays revealed that recovered HMGA2 expression eliminated the inhibitory effects on cell proliferation (Fig. 5B, P<0.05) and invasion (Fig. 5C, P<0.05) induced by miR-599 overexpression in A498 and 786-O cells. Therefore, miR-599 inhibits cell proliferation and invasion of ccRCC partly by inhibiting the HMGA2 expression.