All 30 samples of ccRCC tissues and paired adjacent non-tumor tissues used in the present study were obtained from 30 patients who had undergone radical nephrectomy at the Department of Urology of Renmin Hospital, Hubei University of Medicine (Hubei, China) between January 2009 and February 2011. Patients were selected on the basis of the following inclusion and exclusion criteria: i) Definite pathological diagnosis of ccRCC according to the World Health Organization criteria (20); ii) suitable formalin-fixed, paraffin-embedded tissue specimens; iii) complete clinicopathological data. None of the patients received radiotherapy or chemotherapy prior to radical nephrectomy. Following surgical resection, specimens were immediately immersed in RNAlater^® (Qiagen GmbH, Hilden, Germany) for 30 min and then transferred into liquid nitrogen for cryopreservation until use. The clinicopathological features of the patients are presented in Table I. Follow-up of patients was completed in May 2015 and the median observation time was 30 months. All specimens were collected on the basis of their availability for research and following a protocol approved by the Medical Ethics committee of the Renmin Hospital, Hubei University of Medicine (Hubei, China). Written consent was obtained from all patients participating in the study.

Human ccRCC cell lines (786-O, 769-P, CaKi-1 and RLC-310) and a normal immortalized human proximal tubule epithelial cell line (HK-2) were obtained from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in RPMI-1640 medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) in a 100% humidified atmosphere of 5% CO₂/95% air at 37°C.

Total RNA from 30 cases of fresh ccRCC tissues and cell lines were extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was detected and quantified using a NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). Subsequently, RNA samples were reverse-transcribed into complementary first-strand DNA using a High-Capacity RNA-to-cDNA kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The qPCR reaction was then performed on the LightCycler^® 480 Real-Time PCR system (Roche Applied Science, Penzberg, Germany) using the SYBR^® Select Master Mix (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The reaction conditions for PCR were as follows: 95°C for 20 min, followed by 40 cycles of 95°C for 10 sec, 55°C for 30 sec and 72°C for 30 sec. The specific primers for human LOC389332 were as follows: Forward, 5′-GCGCTCGTCGTCCTCTTCATCG-3′ and reverse, 5′-CGTGGCTCAGTCCCAAGCTACACC-3′. The GAPDH gene was used as an internal control, and the primers for GAPDH were: Forward, 5′-GGCACCACACCTTCTACAATGAG-3′ and reverse, 5′-GGATAGCACAGCCTGGATAGCA-3′. All primers were obtained from Invitrogen (Carlsbad, CA, USA). The relative expression levels of LOC389332 in ccRCC tissues and cell lines were analyzed using the 2^−ΔΔCq method (21).

As reduction in the expression of LOC389332 in ccRCC tissues and cells was observed, a gain-of-function study was performed by inducing the overexpression of LOC389332 to identify its function in the 786-O and 769-P cell lines. An LOC389332 overexpression plasmid (pcDNA3.1-LOC389332) was purchased from Thermo Fisher Scientific, Inc. 786-O and 769-P cells were treated with the indicated amounts of pcDNA3.1-LOC389332 plasmid using Lipofectamine^® 2000 reagent (Thermo Fisher Scientific, Inc.). The empty vector pcDNA3.1- was used as a negative control.

A scratch wound assay was applied to evaluate the ability of 786-O and 769-P cells to migrate in vitro. Cells (~2×10⁶/well) were seeded into 6-well plates and grown to 70% confluence ~24 h prior to infection. Following 6 h of transfection, similar-sized wounds were generated to the cell monolayers using sterile 100-µl pipette tips. Following three washes with phosphate-buffered saline to remove cell debris, cells were cultured in the incubator at 37°C. In each sample, images of the same area were acquired with a Leica DM LB2 microscope digital camera system (Leica Microsystems GmbH, Wetzlar, Germany) at 0 and 12 h after the wounds were made to determine the amount of wound closure. The data was calculated using the software program MIAS-2000 (Leica Microsystems GmbH). This experiment was performed in triplicate and repeated at least three times.

786-O and 769-P cell proliferation was assessed using an MTT assay. Transfected cells were seeded onto each well of 96-well plates at a density of 5×10³ cells per well. Following 0, 24, 48, 72 and 96 h of transfection, 15 µl of MTT (5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA) was added to each well and plates were incubated for 4 h at room temperature. Subsequently, 130 µl dimethyl sulfoxide was added to each well and wells were shaken for 10 min at 37°C to solubilize the formazan crystals. The absorbance of 786-O and 769-P cells was measured at 490 nm using a Bio-Rad model 680 microplate reader (Bio-Rad Laboratories Inc., Hercules, CA, USA). Results from the MTT assay are presented as the mean of at least three independent experiments.

Statistical analysis was performed with SPSS 16.0 (SPSS, Inc., Chicago, IL, USA). Values are expressed as the mean ± standard deviation. To compare LOC389332 expression levels in ccRCC tissues vs. matched tumor normal tissues, a paired Student's t-test. was used. The χ² test was applied to compare the levels of LOC389332 expression with various clinicopathological parameters. The results of the MTT assay were analyzed using one-way analysis of variance. Overall survival curves were generated using the Kaplan-Meier method. P<0.05 was considered to indicate a statistically significant difference.

Previous lncRNA expression profiling of ccRCC indicated that LOC389332 was significantly downregulated in RCC tissues (18,19). To confirm that LOC389332 expression is decreased in ccRCC, RT-qPCR was performed to quantify LOC389332 expression in the 30 ccRCC tissues and ccRCC cell lines. The results demonstrated that LOC389332 expression was significantly lower in ccRCC tissues compared with matched adjacent normal tissues (P<0.05; Fig. 1A). In addition, LOC389332 expression was significantly downregulated in 786-O, 769-P, CaKi-1 and RLC-310 cell lines, compared with that in the normal HK-2 cell line (P<0.05; Fig. 1B). These results suggested that LOC389332 expression is suppressed in ccRCC.

To evaluate the association between LOC389332 expression and the patients' clinicopathological features, the 30 ccRCC samples were divided into low (n=21) and high (n=9) LOC389332 expression groups based on the median value of relative LOC389332 expression. As presented in Table I, downregulation of LOC389332 expression was correlated with tumor American Joint Commission on Cancer (AJCC) stage (22) (P=0.001), Fuhrman grade (23) (P=0.001) and lymph node metastasis (P<0.001). However, no correlation was detected between LOC389332 expression and patient gender (P=1.000), age (P=0.418) or tumor size (P=0.100). These results demonstrated that decreased LOC389332 expression was associated with ccRCC progression and development.

Kaplan-Meier analysis revealed that the overall survival rate of the low LOC389332 expression group was significantly lower than the overall survival of the high LOC389332 expression group (P=0.001; Fig. 2). The 3-year survival rate for ccRCC patients with low LOC389332 expression was 54% compared with 83% in patients with high LOC389332 expression (P<0.05).

To identify the biological functions of LOC389332 on ccRCC cells in vitro, transfection of pcDNA3.1-LOC389332 overexpression plasmid were applied to restore LOC389332 expression in 786-O and 769-P cells. RT-qPCR analysis revealed that LOC389332 expression in 786-O and 769-P cells transfected with the pcDNA3.1-LOC389332 plasmid was significantly upregulated compared with that in the pcDNA3.1-empty vector group (Fig. 3A). The MTT assay revealed that the proliferation of the 786-O and 769-P cell lines was significantly decreased in the pcDNA3.1-LOC389332 groups compared with that in the empty vector-transfected groups (Fig. 3B).

The effect of LOC389332 on ccRCC cell migration was observed using a scratch wound assay. Compared with the pcDNA3.1-empty vector group, overexpression of LOC389332 significantly suppressed the migration capacity of 786-O and 769-P cells (P=0.002 in each; Fig. 4), suggesting that ectopic overexpression of LOC389332 impairs the migratory capacity of ccRCC cells.