The present study was approved by the ethics committee of the First Affiliated Hospital of Harbin Medical University (Harbin, China). All RCC tissues and paired adjacent normal tissues used in the present study were collected from The Department of Urology of The First Affiliated Hospital of Harbin Medical University. All specimens were obtained on the basis of their availability for investigation purpose and under a protocol approved by the local medical ethics committee. Written informed consent was obtained from all patients involved in the present study. All tissue samples were reviewed and classified using hematoxylin and eosin staining and the 2009 American Joint Committee on Cancer staging system (18). The clinicopathological information of the patients is presented in Table I. The people were all renal cell (RCC) carcinoma patients. They could improve prognosis after nephrectomy. A total of 60 fresh RCC and adjacent normal tissue samples, located 2.0 cm from the visible RCC lesions, were obtained from patients with RCC by nephrectomy. The samples were immersed in RNAlater (Qiagen, Hilden, Germany) for 30 min and subsequently stored at −80°C. The total RNA of each sample was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. All isolated RNA was quantified using a Nano-Drop^® spectrophotometer. Only RNA samples with 260/280 ratios of 1.8–2.0 were used for further investigation.

CAKI- 2, 769- P, ACHN and 786-O renal carcinoma cell lines were obtained from the laboratory of the Institute of Urology of Shenzhen PKU-HKUST Medical Center (Shenzhen, China). Human embryonic kidney 293T cells were purchased from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO₂. First-strand cDNA was synthesized using oligo-dT primers (cat. no. K1622; Fermentas, Waltham, MA, USA). The primer sequences specific for human TRIM52-AS1 were as follows: Forward 5′-AGA GCA AGG ACT GTA TGT GTTC-3′ and reverse 5′-CTG GAG TGG CAG AAG TAAGG-3′; with human GAPDH as an internal control, the primer sequences for GAPDH were forward 5′-AGT GGC AAA GTG GAG ATT -3′ and reverse 5′-GTG GAG TCA TAC TGG AACA-3′. RT-qPCR was performed using a SYBR^® Premix EX Taq™ II PCR kit (cat. no. RR820A; Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol, on a Roche Light-cycler 480 Real-Time PCR System (Roche Diagnostics). Data were calculated according to the Applied Biosystems comparative quantification method (19).

Cells of the human renal carcinoma cell lines, ACHN and 786-O, were purchased from the American Type Culture Collection (Manassas, VA, USA). TRIM52-AS1 small hairpin shRNA (target sequence, 5′-GCA CAG AGC AAG GAC UGU AUG UGU U-3′) were purchased from Genechem (Shanghai, China). The TRIM52-AS1 overexpression plasmid, TRIM52-AS1-pcDNA3.1+, was synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). Approximately 400,000 renal cancer cells were cultured in six- well plates. The ACHN and 786-O cells were transfected with the TRIM52-AS1 shRNA (200 pmol/well) or TRIM52-AS1-pcDNA3.1+ (4 µg/well) vector using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.).

Cell proliferation was determined using a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich) assay, performed according to the manufacturer's protocol. Briefly, the cells (~5×10³ cells) were seeded into a 96-well culture plate 24 h prior to trans-fection with the TRIM52-AS1 shRNA (200 pmol/well) or TRIM52-AS1-pcDNA3.1+ (4 µg/well)vector. At 0, 24, 48 or 72 h post-transfection, 20 µl MTT (5 mg/ml) was added to each well, and the plates were incubated for 4 h at 37°C. Subsequently, the MTT medium mixtures were discarded, and 150 µl dimethyl sulfoxide was added to each well, and agitated for 10 min at room temperature to solubilize the crystals. The results were measured at a wavelength of 490 nm (with 630 nm as the reference wavelength) using an ELISA microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Assays were repeated at least three times.

A wound scratch assay was used to assess the migratory ability of the 786-O and ACHN RCC cells in vitro. For the assay, ~5×10⁶ cells were seeded per 6-well dish and were transfected with TRIM52-AS1 shRNA (100 pmol) or TRIM52-AS1-pcDNA3.1+ (4 µg) after 24 h using Lipofectamine 2000. At 6 h post-transfection, a vertical horizontal wound was made in the cell layer using a sterile 10 µl pipette tip, and markers were included to allow observation of cells at the same point. The cells were then rinsed with phosphate-buffered saline (PBS) and cultured in an incubator at 37°C. Images of the wounds were captured with a digital camera system (C3040-AD6; Olympus Corporation, Tokyo, Japan) 0 and 24 h following creation of the wounds at the same points. The wound widths (lm) were measured using a standard caliper (Caliper; PerkinElmer, Inc., Waltham, MA, USA). The experiments were performed in triplicate and repeated at least three times.

The extent of apoptosis was evaluated using an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) detection kit (Invitrogen; Thermo Fisher Scientific, Inc.). Following transfection of the ACHN and 786-O cells with the TRIM52-AS1 shRNA (200 pmol/well) or TRIM52-AS1-pcDNA3.1+ (4 µg/well) vector, the cells were collected, washed twice with pre-chilled PBS and re-suspended in 1X binding buffer (pH 7.4; 10 mmol/l HEPES, 140 mmol/l NaCl, 5 mmol/l CaCl₂; Invitrogen; Thermo Fisher Scientific, Inc.), 48 h post-treatment. The aliquots were mixed with 10 µl annexin V-FITC and 10 µl PI at room temperature for 15 min. The apoptosis assay was performed using a flow cytometer (EPICS Xl-4, Beckman, CA, USA). Each experiment was performed at least three times.

Statistical analysis was performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). Statistical significance was determined using Student's t-test. For comparison of the expression levels of TRIM52-AS1 in matched tumor, vs. normal samples, a paired t-test was used. Data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

A previous study showed TRIM52-AS1 to be downregulated in RCC tissues, as determined by lncRNAs expression profiling (16,17). To confirm the result of sequencing, RT-qPCR was used to quantify the expression levels of TRIM52-AS1 in 60 matched RCC tissue samples and adjacent normal tissue samples. The relative expression of TRIM52-AS1 [log2 (N/T)] is shown in Fig. 1A. The expression of TRIM52-AS1 in the RCC tissues was significantly lower, compared with that in the adjacent normal tissues. Furthermore, compared with normal kidney 293T cells, TRIM52-AS1 was also downregulated in the 786-O and ACHN cell lines (P<0.05; Fig. 1B). These results suggested that TRIM52-AS1 may act as a tumor suppressor gene in RCC.

To analyze the function of TRIM52-AS1 in RCC using an MTT assay, either TRIM52-AS1 shRNA or the TRIM52-AS1-pcDNA3.1+ vector were transfected into the 786-O and ACHN cell lines. The optical density (OD) values of the TRIM52-AS1 shRNA or the TRIM52-AS1-pcDNA3.1+ vector-treated groups were measured at a wavelength of 490 nm (620 nm reference wavelength) using an ELISA microplate reader at 0, 24, 48 and 72 h post-transfection. The results demonstrated that the relative proliferation rates of the TRIM52-AS1 shRNA-transfected 786-O cell line were significantly increased by 42.8% (24 h), 31.2% (48 h) and 55.2% (72 h), whereas proliferation rates in the TRIM52-AS1-pcDNA3.1+ vector-transfected group were significantly decreased by 16.8% (24 h), 15.2% (48 h) and 20.4% (72 h; Fig. 2A and B); The relative cell proliferation rates in the TRIM52-AS1 shRNA-transfected ACHN cell line were significantly increased, by 14.8% (24 h), 17.3% (48 h) and 29.2% (72 h), whereas proliferation rates in the TRIM52-AS1-pcDNA3.1+ vector-transfected group were significantly decreased by 21.5% (24 h), 30.6% (48 h) and 54.7% (72 h; Fig. 2C and D). These results indicated that the downregulation of TRIM52-AS1 had a negative effect on cellular proliferation in RCC.

The effects of TRIM52-AS1 on cellular migration in RCC cells were observed using a wound scratch assay. As shown in Fig. 3, interference of the expression of TRIM52-AS1 increased the rate of migration, whereas the overexpression of TRIM52-AS1 decreased migration rate (P<0.05). The results demonstrated that the wound widths in the group of cells transfected with the TRIM52-AS1-pcDNA3.1+ vector were markedly wider than those in the TRIM52-AS1 shRNA group, which indicated that downregulation of TRIM52-AS1 inhibited the migration of RCC cells.

LncRNAs have been reported to be important in cell apoptosis, particularly in the escape of cancer cells from apoptosis (20–22). To determine the impact of TRIM52-AS1 on RCC cell apoptosis, flow cytometry was performed to detect the rate of apoptosis in the cells. As shown in Fig. 4, the apoptotic rates of the 786-O cells transfected with the TRIM52-AS1 shRNA and TRIM52-AS1-pcDNA3.1+ vector were 7.5 and 25.3%, respectively, and the apoptotic rates of the ACHN cells were 5.1 and 15.3%, respectively, which demonstrated that the downregulation of TRIM52-AS1 promoted RCC cell apoptosis.