The normal human hepatocyte cell line L02 and human liver cancer cell line HepG2 were purchased from the Cell Bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The two cell lines were cultured in Dulbecco's Modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), 100 IU/ml penicillin and 100 µg/ml streptomycin) in 5% CO₂ at 37°C, for subsequent experimental research.

A total of 26 HBV-related liver cancer and non-HBV infected liver cancer tissues were obtained from patients who underwent radical resections for primary liver cancer in Nanjing Drum Tower Hospital (Nanjing University, Nanjing, China) during April 2014 to March 2015. These liver cancer samples were from 17 males and 9 females (37-80 years old). All patients were diagnosed by histopathological examination and the detection of HBV markers. In the subsequent experiments, all patients were separated into two groups based on lncRNA-ATB expression, and the cut-off was the 50th percentile. Tissues following the resection procedures were immediately placed in liquid nitrogen, then stored at −80°C. The present study was approved by the Ethics Committee of Nanjing Drum Tower Hospital and written informed consent was provided by all patients.

For the development of lentiviral vectors expressing the HBx gene, HBx cDNA was amplified by polymerase chain reaction (PCR) and subcloned into the lentiviral vector pHBLV-CMVIE-ZsGreen-T2A-Puro using the one step directed cloning kit (Hanbio Biotechnology Co., Ltd., Shanghai, China); empty vector and non-transfected cells served as the control. The restriction enzyme used for HBx cDNA and vector were EcoRI/XhoI and XbaI/BamHI, respectively (Takara Biotechnology Co., Ltd., Dalian, China). The PCR procedure includes: Pre-denaturation at 95°C for 5 min, 95°C for 45 sec, 60°C for 45 sec, 72°C for 1 min (35 cycles) and finally the last cycle 72°C for 10 min, using an ABI StepOne Plus system (Applied Biosystems; Thermo Fisher Scientific, Inc.). To produce lentiviruses containing the HBx gene, the present study transfected 293 cells (Hanbio Biotechnology Co., Ltd.) with the resulting vector described above, pSPAX2 and pMD2G (Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols (23). Infectious lentiviruses were harvested at 48 and 72 h post-transfection and filtered through 0.45 µm polyvinylidene fluoride (PVDF) filters, then centrifuged at 4°C, 72,000 × g/min for 120 min in a 40 ml ultracentrifuge tube, dissolved in DMEM and stored in liquid nitrogen. These lentiviruses were termed LV-control and LV- HBx, respectively. The two lentiviral vectors encoded green fluorescent protein.

The respective recombinant lentiviruses were added to HepG2 cells plated in a 6-well plate with a MOI of 20 to obtain cell lines stably expressing HBx. The supernatant was replaced with complete culture media (DMEM with 10% fetal bovine serum) following 24 h. Cells stably expressing HBx were screened out following the addition of 2 µg/ml puromycin into the media. Reverse transcription-quantitative PCR (RT-qPCR) and western blotting were performed to confirm the expression of HBx in the infected cells following infection.

The induction of cell autophagy was conducted according to Klionsky et al (24). The cell medium of each group was refreshed when the 6-well plate was covered with cells at 85% confluence; the control cells were cultured with fresh DMEM, and the experimental cells were washed three times with PBS to remove the residual medium and then cultured with 1X Earle's balanced salt solution (EBSS; Invitrogen; Thermo Fisher Scientific, Inc.) instead of DMEM, to generate a starvation environment with sugar and amino acid deficiency, and consequently induce autophagy. Cells were then incubated at 37°C and 5% CO₂ for 8 h and the optimal duration for the induction of autophagy selected in this experiment was 4 h post-starvation. Following the successful establishment of the model, total RNA and protein in each group of cells were extracted for subsequent analyses.

Small interfering (si)RNAs against lncRNA-ATB and TGF-β (40 nmol/l) were transfected into HepG2 and HBx-HepG2 cells respectively, which were plated in a 6-well plate at a density of 50%, using Lipofectamine 3000 according to manufacturer's instructions. Scramble-control siRNA was used as the control, which was transfected into the cells in the same manner as si-ATB and si-TGF-β transfection. The catalogue numbers of control/si-TGF-β/si-ATB are siN05815122147, siB09212165524 and siB160908101333, respectively (Guangzhou RiboBio Co., Ltd.). RT-qPCR confirmed the knockdown effect in HepG2 and HBx-HepG2 cells at 48 h post-transfection.

Total RNA was isolated from cultured cells or tumor tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The first-strand cDNA was synthesized using PrimeScript™ RT Master Mix (Takara Biotechnology Co., Ltd.); the conditions for RT were: 37°C for 20 min and then heated at 85°C for 15 sec. cDNAs could be stored at -20°C. qPCR was conducted with mixed cDNAs, gene primers and SYBR Green PCR master mix (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer's protocols, while the RNA expression levels were measured using an ABI StepOne Plus system (Applied Biosystems; Thermo Fisher Scientific, Inc.). QPCR reactions were performed in triplicate, while β-actin and 18S rRNA were selected as the internal controls. The primers utilized for qPCR were as follows: lncRNA-ATB forward, 5′-TCTGGCTGAGGCTGGTTGAC-3′, reverse, 5′-ATCTCTGGGTGCTGGTGAAGG-3′; HBx forward, 5′-CCCGTCTGTGCCTTCTCATC-3′, reverse, 5′-GTATGCCTC-AAGGTCGGTCG-3′; β-actin forward, 5′-GGGAAATCGTGCGTGACATTAAG-3′, reverse, 5′-TGTGTTGGCGTACAGGTCTTTG-3′; Beclin-1 forward, 5′-CAGGAGAGACCCAGGAGGAA-3′, reverse, 5′-GCTGTTGGCACTTTCTGTGG3′; microtubule-associated proteins 1A/1B light chain 3B (LC3b) forward, 5′-CCGCACCTTCGAACAAAGAG-3′, reverse, 5′-TTGAGCTGTAAGCGCCTTC-T-3′; sequestosome 1 (SQSTM1) forward, 5′-TCTGGCTGAGGCTGGTTGAC-3′, reverse, 5′-ATCT-CTGGGTGCTGGTGAAGG-3′ and TGF-β forward, 5′-CACCATAAAGACAGGAACCTG-3′, reverse, 5′-GGAGGTGCCATCAATACCTGC3′. The relative expression of RNAs was calculated using the comparative 2^−ΔΔCq method (25,26).

Lysis buffer was used for the extraction of total proteins of cells and tissues, which were quantified using a Bicinchoninic Acid kit (Nanjing KeyGEN Biotech Co., Ltd., Nanjing, China). Equal quantities of proteins (4 mg/ml) were separated by SDS-PAGE (with 30% acrylamide), then transferred onto PVDF membranes. Following incubation with antibodies (1:1,000) specific for HBx (ab39716; Abcam, Cambridge, UK), LC3b (cat. no. 3868; Cell Signaling Technology, Inc., Danvers, MA, USA), p62 (cat. no. 23214; Cell Signaling Technology, Inc.), Beclin-1 (cat. no. 4122; Cell Signaling Technology, Inc.), TGF-β (ab186838; Abcam) and β-actin (ab179467; Abcam) for 12 h at 4°C; then the blots were incubated with goat anti-rabbit (for HBx, LC3b and p62) or anti-mouse (for TGF-β, β-actin and Beclin-1) IgG-horseradish peroxidase-conjugated antibodies (1:5,000; Nanjing KeyGEN Biotech Co., Ltd.) for 2 h at room temperature. The proteins were semi-quantified by chemiluminescence method (Tanon Science & Technology Co., Ltd., Shanghai, China) and analyzed using ImageJ software (Version 1.5.1; National Institutes of Health, Bethesda, MD, USA).

A Transwell assay was used to determine cell invasion as described previously (27). Cells (~5×10⁴) were resuspended in 200 µl serum-free medium and added to the upper layer of the Transwell chamber, while 500 µl of complete medium was added into the lower chamber. Following 48 h of culture at 37°C, the medium was discarded and noninvaded cells were gently removed with a cotton swab and rinsed three times with PBS. The remaining cells were then fixed using 3.7% paraformaldehyde at room temperature for 20 min. The membranes were stained with 0.1% crystal violet for 30 min at room temperature, and cell invasive ability was analyzed by counting the number of stained cells in random fields of view under a 400-fold inverted biological microscope.

Cell migration ability was evaluated using a scratch assay. A 200-µl pipette tip was used to scratch a straight wound in cells seeded in 6-well plates and then cultured in serum-free medium for 24 h at 37°C. Images of the wound width were then captured at 0 and 24 h and compared under a 5-fold inverted biological microscope. All experiments were independently repeated in triplicate.

SPSS 19.0 software (IBM Corp., Armonk, NY, USA) was used for statistical analysis. Each group of experiments was repeated at least three times, and data are expressed as the mean ± standard deviation. The χ² test was used to assess the association between lncRNA-ATB expression and the clinical characteristics of patients. A two-tailed Student's t-test was used for the comparison of independent variables. One-way analysis of variance was used for the comparison of multiple groups, followed by a Student-Newman-Keuls post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

To clarify the role of lncRNA-ATB in primary liver cancer and whether HBx regulated lncRNA-ATB, the present study selected 26 tumor tissues from patients with radical hepatectomy and detected the expression of lncRNA-ATB by RT-qPCR. The association between lncRNA-ATB content and the clinicopathological characteristics of liver cancer, including the status of HBV infection, was then evaluated. As presented in Fig. 1, lncRNA-ATB was significantly associated with HBV. In addition, a significant association was detected between more advanced TNM stage and a higher expression levels of lncRNA-ATB, irrespective of patient age, gender, tumor size, liver cirrhosis and histological differentiation (Table I). These results suggested that increased expression of lncRNA-ATB in primary liver cancer may be associated with HBV infection and advanced tumor development.

Previous studies have revealed that lncRNA-ATB can promote the invasion and migration of various tumor cells including primary liver cancer by activating the EMT and STAT3 pathways, while lncRNA-ATB could induced by TGF-β (18,28-30). However, whether abnormal expression of lncRNA-ATB is associated with the invasive and migration abilities of HBV-associated liver cancer cells remains unknown. To clarify this mechanism, the present study constructed an HBx lentivirus transfected HepG2 cell line (HBx-HepG2) which stably expressed HBx protein. The results of western blotting and RT-qPCR revealed that the levels of HBx protein and mRNA were markedly increased in HepG2 cells following transfection, while the expression of HBx in the blank control and empty plasmid groups (con-HepG2) was notable reduced (Fig. 2A and B).

To further investigate the effects of HBx on lncRNA-ATB in HBx-HepG2 cells, the present study compared the expression of lncRNA-ATB in L02, HepG2, con-HepG2 and HBx-HepG2 cell lines. As presented in Fig. 2C, the content of lncRNA-ATB in HBx-HepG2 cells was significantly increased following transfection with HBx lentivirus compared with HepG2 cells. The results were similar in HepG2 and con-HepG2 cells. In addition, the levels of TGF-β mRNA expression, which has been reported to be an inducer of lncRNA-ATB expression, were evaluated; a significant increase in HBx-HepG2 cells was observed compared with HepG2 cells (Fig. 2D). These results suggested that HBx may promote the expression of lncRNA-ATB in HepG2 cells by upregulating the expression of TGF-β.

As aforementioned, alterations in cell behaviors, including metabolism, secretion or degradation of functional proteins, is one of the most critical underlying mechanisms as to how HBx leads to the development of primary liver cancer (5,31). In recent years, autophagy has been reported as an essential mechanism for regulating the intracellular environment (20,32). Whether HBx can affect the expression of intracellular lncRNA-ATB by mediating the autophagy of liver cancer cells is unclear. To investigate this, the present study detected the level of autophagy in HepG2 and HBx-HepG2 cells. As presented in Fig. 3A and B, the expression of autophagy-associated markers SQSTM-1 and LC3 in HBx-HepG2 cells were significantly increased following HBx transfection, while Beclin-1, which reflects the successful synthesis of autophagic precursors, exhibited a significant decrease compared with in HepG2 cells. HBx-HepG2 cells exhibited a marked increase in the expression of p62 compared with in HepG2 cells. These results indicated that HBx could promote autophagy in HepG2.

To further clarify whether lncRNA-ATB expression levels in HBx-HepG2 are associated with high levels of autophagy, the present study designed starvation-induced autophagy models using EBSS, which has been widely used for the induction of autophagy (33,34). Alterations in autophagy-associated proteins in each group were detected once cells were cultured with serum-free essential culture medium EBSS for 4 h. The expression levels of Beclin-1 and LC3 were significantly increased in HepG2 and HBx-HepG2 cells following 4 h of starvation (Fig. 3C-E). The increase in LC3 content and LC3 II/LC3 I indicated an increase in autophagy, while the upregulated Beclin-1 suggests that autophagy may occur rapidly and autophagic precursor protein may be synthesized in marked quantities.

Following the successful induction of autophagy, the content of lncRNA-ATB in HepG2 and HBx-HepG2 cells increased significantly at 4 h compared with 0 h of starvation (Fig. 4A). Analysis of the lncRNA-ATB inducer, TGF-β, revealed that the mRNA and protein expression levels of TGF-β were upregulated in the two cell lines at 4 h of starvation than at 0 h (Fig. 4B-D). These results indicated that starvation-induced autophagy could significantly increase the levels of lncRNA-ATB and TGF-β in HepG2 cells transfected with HBx.

The aforementioned findings indicated an association between autophagy and TGF-β/lncRNA-ATB expression in HBx-HepG2 cells. To further verify this association, the present study employed siRNAs against lncRNA-ATB and TGF-β to observe their effects on cell autophagy following knockdown of lncRNA-ATB/TGF-β. The results of RT-qPCR demonstrated that the expression of target RNA in cells was significantly inhibited following siRNA-ATB or siRNA-TGF-β transfection in HepG2 and HBx-HepG2 cells compared with the control, indicating successful transfection (Fig. 5A and B). In addition, the expression of lncRNA-ATB was significantly decreased following knockdown of TGF-β compared with the control (Fig. 5C). Conversely, the expression levels of TGF-β were markedly altered following the knockdown of lncRNA-ATB (data not shown), which suggested that TGF-β also regulates the expression of lncRNA-ATB in HBx-HepG2 cells.

Further investigation demonstrated that LC3 expression was suppressed following the knockdown of lncRNA-ATB or TGF-β in HepG2 cells; however, these observations were not noted in HBx-HepG2 cells (Fig. 5D-G). These results demonstrated that, at least in part, autophagy was inhibited in the case of lncRNA-ATB knockdown in liver cancer cells, while the expression of HBx could partially alleviate this inhibition.

To further detect the association between HBx-induced autophagy and lncRNA-ATB, the present study determined lncRNA-ATB expression in starvation-induced autophagy and the suppression of TGF-β in HepG2 and HBx-HepG2 cells. The mRNA levels of the autophagy-associated protein LC3b were significantly increased following starvation for 4 h, while such an increase was observed in HBx-HepG2 and HepG2 cells transfected with si-TGF-β (Fig. 6A and B). Combined with previous experiments, this suggested that HBx-induced autophagy could promote the expression of TGF-β but was not dependent on it. The present study then investigated the expression levels of lncRNA-ATB under these conditions. As presented in Fig. 6C and D, si-TGF-β significantly inhibited the expression of lncRNA-ATB compared with the control at 0 h, but was significantly upregulated by starvation-induced autophagy in HepG2 and HBx-HepG2 cells. Thus, it was concluded that HBx-induced autophagy led to the abnormal expression of TGF-β, and upregulated that of lncRNA-ATB in liver cancer cells.

It has been reported that the invasion and migration abilities of HBV-associated liver cancer cells were greater than normal hepatocyte cell lines (35,36); however, whether lncRNAs participate in the invasion and migration of HBV-associated primary liver cancer is unclear. In the present study, stably transfected HBx-vector significantly promoted cell invasion and migration compared with HepG2 cells (Fig. 7A-D). A scratch-wound assay was then conducted to investigate whether TGF-β or lncRNA-ATB affected the migration ability of HBx-HepG2 cells. As presented in Fig. 7E-H, the migration ability of HBx-HepG2 cells was significantly inhibited when TGF-β was suppressed compared with the control group, which was not apparent in HepG2 cells. In addition, such inhibition of migration was significant in the two types of cells transfected with siRNA-ATB (Fig. 7I-L). Therefore, it was concluded that the invasion and migration of HBx-HepG2 may be associated with the expression of lncRNA-ATB. Abnormal expression of lncRNA-ATB may be an underlying mechanism of enhanced invasion and migration of HBV-associated liver cancer cells.