Tumor tissues and paired noncancerous tissues were collected from 30 patients with HCC admitted to the Department of Radiology, Jingzhou Central Hospital (Jingzhou, China) between 2010 and 2012. Ethical approval for the study was provided by the Independent Ethics Committee of Jingzhou Central Hospital. Informed and written consent was obtained from all patients or their advisers according to the ethics committee guidelines.

HEK293T, HCC, HepG2, HuH7, BEL-7404, SMMC-7721, MHCC-97L and MHCC-97H cells were obtained from the Cell Bank of Shanghai Biology Institute, Chinese Academy of Science (Shanghai, China). All culture media were supplemented with 10% fetal bovine serum, 100 mg/ml penicillin G and 50 µg/ml streptomycin (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA). HEK293T, HepG2, HuH7, BEL-7404, MHCC-97L and MHCC-97H cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies; Thermo Fisher Scientific, Inc.) and the SMMC-7721 cells were cultured in Roswell Park Memorial Institute 1640 medium (Life Technologies; Thermo Fisher Scientific, Inc.). All cells were maintained at 37°C in 5% CO₂.

pLKO.1, psPAX2 and pMD2.G were purchased from Addgene, Inc. (Cambridge, MA, USA). Three small hairpin RNAs (shRNAs; Generay Biotech Co., Ltd., Shanghai, China) targeting human RTKN2 mRNA were cloned into a lentiviral vector (PLKO.1). A non-specific scramble shRNA sequence (CCT AAG GTT AAG TCG CCC TCG) was used as negative control. The constructs were then transfected into HEK293T cells with lentiviral packaging vectors (psPAX2 and pMD2.G) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Viruses were collected 48 h subsequent to transfection and used to infect HepG2 and BEL-7404 cells. After 48 h, the cells were processed for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. The shRNA target sequences for RTKN2 were listed in Table I.

Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. cDNA was synthesized using 1 µg total RNA, 0.5 mM dNTPs, 2.5 µM oligo-dT primer (for mature mRNAs), 1 U/µl RiboLock RNase Inhibitor and 10 U/µl M-MuLV Reverse Transcriptase (all from Thermo Fisher Scientific, Inc.). The cDNA synthesis was performed for 1 h at 37°C and the reaction was stopped by 10 min incubation at 70°C.

RT-qPCR was performed in an Applied Biosystems 7300 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the Absolute Blue qPCR SYBR Green Low ROX mix (Thermo Fisher Scientific, Inc.). For each reaction 5 µl cDNA template, 1 µl primer pairs (10 µM) and 10 µl of the qPCR SYBR mixture to a final reaction volume of 20 µl were used. All reactions were conducted using the following cycling parameters: 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 45 sec, and a final extension step at 72°C for 5 min. Relative expression levels at the tested experimental conditions were calculated within each independent experiment using the using the ΔΔCq method (13). All data represent the average of three replicates. The quality of the PCR product was monitored using post-PCR melt curve analysis. The sequences of the primer pairs used are as follows: RTKN2 (NM_001282941.1), F 5′-ACAGTTCGCGTTGGAGATGGAG-3′ and R 5′-GTCGAGCATTGCACACCATGAG-3′; glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (NM_001256799.1), F 5′-CACCCACTCCTCCACCTTTG-3′ and R 5′-CCACCACCCTGTTGCTGTAG-3′.

Treated and untreated HepG2 and BEL-7404 cells were washed twice with phosphate-buffered saline (PBS) and lysed in ice-cold radioimmunoprecipitation assay buffer (JRDUN Biotechnology Co., Ltd., Shanghai, China) with freshly added 0.01% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and incubated on ice for 30 min. Subsequent to centrifugation, supernatants were collected and stored at −80°C until required for use. For western blot analysis, the cell lysates were run on a 10–15% sodium dodecyl sulfate-polyacrylimide gel electrophoresis gel and transferred electrophoretically to a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). The blots were blocked with 5% skimmed milk with gentle shaking, followed by incubation with primary antibodies. Blots were then incubated with the horseradish peroxidase-conjugated goat anti-mouse (cat. no. A0216; dilution 1:1,000) or goat anti-rabbit (cat. no. A0208; dilution 1:1,000) secondary antibodies (Beyotime Institute of Biotechnology, Shanghai, China) then were visualized using enhanced chemiluminescence (ECL; EMD Millipore). Mouse monoclonal anti-PTKN2 (cat. no. ab118069; dilution 1:200), rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA; cat. no. ab92552; 1:5,000) and mouse monoclonal anti-cyclin-dependent kinase 1 (CDK1; cat. no. ab18; 1:1,000) antibodies were purchased from Abcam (Cambridge, MA, USA). The rabbit monoclonal anti-GAPDH antibody (cat. no. 5174; 1:1,500) was from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cell proliferation was measured using the Cell-Counting Kit-8 (CCK-8) Assay kit (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's instructions. In brief, the treated and untreated HepG2 and BEL-7404 cells were seeded onto 96-well plates. At the indicated time points, CCK-8 solution (10 µl in 100 µl DMEM) was added to each well and incubated for 1 h. Optical density values at a wavelength 450 nm were measured using a microplate reader (model 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA). All experiments were run in triplicate and repeated a minimum of three times.

The cell cycle was analyzed as previously described (14) with slight modifications. Briefly, cells cultured on 60-mm diameter dish were infected with the indicated virus. After 48 h, cells were harvested by trypsinization (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), washed with ice-cold PBS, and fixed with ice-cold 70% ethanol for a minimum of 2 h at −20°C. The fixed cells were washed with PBS, incubated with ribonuclease A (Sigma-Aldrich) and propidium iodide (PI; 0.05 mg/ml, Sigma-Aldrich) at room temperature in the dark for 30 min. DNA content was then analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). The percentage of cells in the G₀/G₁, S and G₂/M phases were determined with FlowJo software version 7.6 (Tree Star, Inc., Ashland, OR, USA). Experiments were performed in triplicate and 3×10⁴ cells were analyzed per sample.

The percentage of cells actively undergoing apoptosis was determined by double staining with annexin V-fluorescein isothiocyanate (FITC; BD Biosciences) and PI (Sigma-Aldrich). Cells were cultured on 60-mm diameter dishes and were infected with the indicated viruses. After 48 h, cells were harvested and then double-labeled with annexin V-FITC and PI as described by the manufacturer. Cells were analyzed using a FACScan flow cytometer.

Invasion chambers (BD Biosciences) were prepared by coating the membranes (8 mm pores) of 24-well inserts with 10 mg/ml Matrigel (BD Biosciences) leaving them to set for 1 h at 37°C. Indicated cells were serum starved for 24 h, then a total of 5×10⁴ cells were added to the upper chamber and suspended in 200 µl DMEM, with 500 µl serum-free DMEM added into the lower chamber. Subsequent to 48 h of incubation, the cells on the upper surface of the filter were completely removed using a cotton bud. The remaining invaded cells were washed with PBS, fixed in 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) and then were stained with 0.2% crystal violet (Beijing Solarbio Science & Technology Co., Ltd.). The invading cells were observed under the microscope (ECLIPSE Ti-S; Nikon Corporation, Tokyo, Japan) at a magnification of ×200 in 10 different fields from each filter. Cells were counted in the central field of triplicate membranes. Experiments were repeated three times.

All data are presented as the mean ± standard deviation. Statistical significance was determined by Student's two-tailed t-test. P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression levels of RTKN2 were compared between HCC tissues and the adjacent normal tissues using RT-qPCR. Overexpression of RTKN2 was identified in 83% (25/30) of the measured HCC tissues. Statistical analysis using Student's t-test demonstrated that the RTKN2 mRNA expression levels were significantly overexpressed in HCC tissues when compared with normal tissues (Fig. 1A; P<0.0001).

In addition, the RNA-sequencing data of the HCC cohort of The Cancer Genome Atlas project (15) was analyzed and it was identified that RTKN2 expression significantly increased in HCC tumor tissues compared with the adjacent tissues of patients (Fig. 1B).

The expression levels of RTKN2 were evaluated using RT-qPCR and western blotting in the following six HCC cell lines: HepG2, HuH7, BEL-7404, SMMC-7721, MHCC-97L and MHCC-97H. Two cell lines, HepG2 and BEL-7404, exhibited higher RTKN2 mRNA and protein expression, while the remaining four cell lines, HuH7, SMMC-7721, MHCC-97 L and MHCC-97H, exhibited reduced mRNA and protein expression (Fig. 2A).

To investigate the functions of RTKN2 on HCC, the HepG2 and MHCC-97H cell lines were selected for further study due to the observed overexpression of RTKN2. Three shRNAs targeting RTKN2 were all able to efficiently suppress endogenous RTKN2 in HepG2 (Fig. 2B) and BEL-7404 cells (Fig. 2C). The RTKN2-Ri-3 shRNA virus was identified to be the most efficient shRNA, thus was stably infected into HepG2 and BEL-7404 cells for further functional analysis.

To examine the effects of RTKN2 downregulation on the proliferation of HCC cells, the proliferation of RTKN2 knockdown cells was assessed using the CCK-8 assay. As presented in Fig. 3A and B, cell growth was markedly impaired in RTKN2-RNAi virus-infected cells compared with wild type cells (WT) and scramble shRNA virus-infected cells (NC). The data indicated a role for RTKN2 in the promotion of HCC cell proliferation.

The possible effect of RTKN2 knockdown on cell cycle progression was then investigated. PI staining and flow cytometry analysis (Fig. 3C) indicated that the population of RTKN2-RNAi HepG2 cells in G₀/G₁ phase was significantly increased by 39.6% (P<0.05), and the population of S phase cells was markedly reduced, by 68.0%, compared with cells infected with scramble shRNA virus-infected cells (NC). Similar results were obtained in BEL-7404 cells (Fig. 3D). These results suggested that RTKN2 promoted G₁/S cell cycle transition in HCC cells.

The expression levels of cell cycle-associated proteins, PCNA and CDK1 were evaluated by western blotting (Fig. 3E and F). Suppression of RTKN2 expression significantly reduced PCNA expression (16), a marker of cell proliferation, by 53.0 and 69.6% in HepG2 and BEL-7404 cells, respectively, compared with corresponding NC cells. The protein level of CDK1, which induces mitosis during cell cycle progression (17), was also reduced in RTKN2 knockdown cells. These results suggested that suppression of RTKN2 expression inhibited cell cycle transition in HCC cells.

To substantiate the role of RTKN2 on cell apoptosis, annexin V/PI staining was performed (Fig. 4). The ratio of HepG2 cells undergoing apoptosis was significantly increased to 29.4±0.66% in RTKN2-RNAi cells, compared with WT (2.5±0.46%) and NC cells (3.53±0.35%) (Fig. 4A). Similar results were observed in BEL-7404 cells (Fig. 4B). These data suggested RTKN2 may serve an anti-apoptotic role in HCC cells.

To investigate whether RTKN2 affected the invasive ability of HCC cells, a Matrigel-coated membrane chamber invasion assay was conducted (Fig. 5). By contrast to control cells (WT and NC), a significant reduction in the invasive ability was observed in RTKN2 knockdown cells. The number of invaded RTKN2-RNAi cells was less than 40% of that of NC cells (HepG2 cells: WT, 61±3; NC, 54±2; RTKN2-RNAi, 21±1. BEL-7404 cells: WT, 66±2; NC, 62±2; RTKN2-RNAi, 22±3). These data suggested that RTKN2 promoted HCC cell invasion.