Human hepatoma cells SK-Hep1 and hepatic cells LO2 (Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences, Shanghai, China) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. All reagents were obtained from Corning Incorporated (Corning, NY, USA).

pHAGE-HBx vector expressing full-length HBx was constructed. The synthesized full-length HBx gene was inserted into the pHAGE vector to generate pHAGE-HBx plasmid. The construct was confirmed by DNA sequencing. LO2 and SK-Hep1 cells were transfected using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) using 2.5 µg plasmids in a 6-well plate, according to the manufacturer's protocol. For stable transfection, LO2 and SK-Hep1 cells were transfected using pHAGE-HBx (or pHAGE-vector) for 48 h and maintained in DMEM containing 2 µg/ml puromycin. After 3–4 weeks of selection, individual colonies were isolated and expanded. The increased expression of target genes in these colonies was confirmed using the reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Total RNA was extracted from HBx transfected or the control cells using an Ultrapure RNA kit (CW0586S; CW Biotech, Beijing, China). A total of 400 ng RNA was transcribed into cDNA using the PrimeScript RT Mastermix (cat.no. RR036Q; Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. The cDNA was amplified using the SYBR-Green PCR Master Mix (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The thermocycling conditions were as follows: Initial denaturation of 95°C for 5 min followed by 40 cycles of 94°C for 15 sec and final extension of 55°C for 1 min. The following primers were used: HBx forward, 5′-actctcagcaatgtcaacg-3′ and reverse, 5′-atttatgcctacagcctcc-3′; GAPDH forward, 5′-tgaaggtcggagtcaacgga-3′ and reverse 5′-cctggaagatggtgatgggat-3′. Reactions were performed in triplicate. Relative expression of HBx was determined using the 2^−ΔΔCq method (22).

Protein lysates from tissues were prepared using a 1% protease and phosphatase inhibitor cocktail [Halt™ Protease and Phosphatase Inhibitor Cocktail, EDTA-Free (100X); cat. no. 78441; Thermo Fisher Scientific, Inc.] in a lysis buffer (4% SDS, 0.1 M dithiothreitol and 0.1 M Tris/HCl, pH 7.6). Cells were lyzed in radioimmunoprecipitation assay buffer (50 mM Tris/HCl, 1% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA and 0.1% SDS) containing a protease and phosphatase inhibitor cocktail (as above). The total protein concentration was measured by BCA assay and samples containing equal amounts of total protein (30 µg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then blocked with 5% non-fat milk in Tris-buffered saline at room temperature for 1 h. Following blocking, membranes were incubated overnight at 4°C with the following primary antibodies: Rabbit anti-WNK1 antibody (1:500; cat. no. 4979S; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-p-PDK1 (Ser²⁴¹) antibody (1:500; cat. no. 3061S; Cell Signaling Technology, Inc.), rabbit anti-PDK1 antibody (1:500; cat. no. 3062S; Cell Signaling Technology, Inc.), anti-p-WNK1 (Thr⁶⁰) antibody (1:200; cat. no. AF4720; R&D Systems, Inc., Minneapolis, MN, USA) and anti-GAPDH antibody (1:2,000; cat. no. TA08; OriGene Technologies, Inc., Beijing, China). Membranes were then washed and incubated with secondary antibodies Goat Anti-Mouse IgG, HRP Conjugated (1:5,000; cat. no. CW0102; CW Biotech, Beijing, China) or Goat Anti-Rabbit IgG, HRP Conjugated (1:5,000; cat. no. CW0103; CW Biotech) at room temperature for 1 h. The protein bands were visualized using enhanced chemiluminescence Western Blotting Substrate (cat. no. 32106; Pierce; Thermo Fisher Scientific, Inc.)

Hepatic cell viability was detected using a Cell Counting kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Cells (5×10³ cells/well in 100 µl medium) were seeded in a 96-well plate. Cells were treated with 0, 5 or 20 µM PDK1 inhibitor II (cat. no. 521276; Merck KGaA, Darmstadt, Germany) in a humidified incubator (37°C, 5% CO₂). Following incubation for the indicated durations, a 1/10 volume of the CCK-8 solution was added to each well and cells were incubated for a further 1 h. Cell viability was determined by measuring the absorbance at 450 nm using a plate reader (Spectramax Paradigm, Molecular Devices, LLC, Sunnyvale, CA, USA).

For the Transwell migration assay, 2×10⁴ cells [suspended in DMEM containing 0.5% fetal bovine serum (FBS)] were trypsinized and plated in the upper chamber of fibronectin-coated plates (8 µm pore filter, Corning Incorporated). The cells were then treated with 0, 5 or 20 µM PDK1 inhibitor II. The lower chamber of the Transwell contained DMEM supplemented with 2.5% FBS. At 20 h, the filters were removed and the cells located on the membrane were fixed with methanol for 2 h. The cells that had migrated to the underside of the membrane were stained using 0.5% crystal violet at room temperature for 1 h and examined under a light microscope (Nikon Eclipse 80i; Nikon Corporation, Tokyo, Japan).

Primary HCC tissue and the paired normal liver tissue specimens were obtained from 21 patients with primary liver cancer who underwent surgical resection at China-Japan Union Hospital of Jilin University (Jilin, China). The samples were collected between March 2012 and February 2015. Of the total 21 patients, 17 (81%) were male and 4 (19%) were female. The median age was 57.8 years (range, 42–73 years). All participants provided written informed consent. Ethical approval was obtained from the research ethics committee of China-Japan Union Hospital of Jilin University.

Data were analyzed using GraphPad Prism (version 6.0) software (GraphPad Software, Inc., La Jolla, CA, USA). The relevant data are expressed as the mean ± standard deviation. Statistical significance between two groups was determined using Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the association between HBx and PDK1, the expression of PDK1 and WNK1 was examined in HBx-overexpressing hepatic cell lines. Hepatic cell lines SK-Hep1 and LO2 were transfected with pHAGE-HBx or pHAGE-vector constructs and the overexpression of HBx in the pHAGE-HBx construct was confirmed using RT-qPCR (data not shown). Expression levels of p-PDK1 (Ser²⁴¹) and p-WNK1 (Thr⁶⁰) were markedly upregulated in HBx-transfected hepatic cells compared with in the control group (Fig. 1A). These results indicate that HBx may affect the phosphorylation of PDK1 and WNK1 in hepatic cells.

SK-Hep1 and LO2 cell lines were employed to establish HBx-stable expression cell lines in order to study the mechanism underlying HBx-mediated regulation of PDK1. Stable expression of HBx by gene transfection was confirmed using RT-qPCR. HepG2.2.15 was used as an HBx-positive control cell line (Fig. 1B). Next, the expression of PDK1 and WNK1 was evaluated in transfected or non-transfected SK-Hep1 and LO2 cell lines treated with 20 µM PDK1 inhibitor for 0, 5, 10, 20, 30 and 60 min (Fig. 1C and D). The phosphorylation of PDK1 and WNK1 were inhibited when SK-Hep1 and LO2 cells were treated with a PDK1 inhibitor (Fig. 1C and D). The effects of the inhibitor were decreased in stable cell lines expressing HBx compared with the control (Fig. 1C and D). These results demonstrate that WNK1 is one of the downstream effectors of PDK1. Additionally, HBx may attenuate the effect of the PDK1 inhibitor on hepatic cells and thus regulate the PDK1/WNK1 signaling pathway.

The LO2-HBx cell line with stable expression of HBx exhibited increased viability compared with the control (data not shown). To investigate how HBx affects hepatic cell viability, a CCK-8 assay was performed on LO2/LO2-HBx and SK-Hep1/SK-Hep1-HBx cells. Hepatic cells were treated with 0, 5, 20 µM PDK1 inhibitor for 72 h (Fig. 2A and B) and with 20 µM PDK1 inhibitor for 0, 24, 48 and 72 h (Fig. 2C and D). The viability of all cells was significantly suppressed at 48 and 72 h, whereas the suppression in the HBx stable-expression cells was weaker compared with the control cells. Thus, the results of the present study suggest that HBx may attenuate the effect of PDK1 inhibitor on the viability of hepatic cells.

To investigate the function of the PDK1/WNK1 signaling pathway in the metastasis of hepatic cells, a Transwell assay was employed to examine the migratory ability of LO2/LO2-HBx (Fig. 3A and B) and SK-Hep1/SK-Hep1-HBx cells (Fig. 3C and D). The results indicated that the migration of cells was gradually decreased in response to increasing doses of the PDK1 inhibitor in LO2/LO2-HBx (Fig. 3B) and SK-Hep1/SK-Hep1-HBx cells (Fig. 3D). No significant differences in the migratory ability were identified between the control and corresponding stable HBx-expressing cells.

Since activation of PDK1 is associated with overexpression of HBx, p-PDK1 expression was evaluated in primary HCC and paired normal liver tissue specimens (Fig. 4A). Increased expression of p-PDK1 was identified in 71% (15/21) of the HCC samples compared with in the corresponding non-tumor livers (Fig. 4B). These results indicate that p-PDK1 may be associated with the development of HBV-associated HCC.