Background
Chronic Hepatitis B virus (HBV) infection is a major risk factor of HCC and is reported to be associated with more than half of HCC cases worldwide [
1,
2]. The HBx, coded by the HBV X gene, is believed to play a vital role in the pathogenesis of HCC by influencing cell cycle, proliferation, and apoptosis at the levels of cell signaling and transcription [
3,
4]. Moreover, HBx also functions as a broad-acting transcriptional activator to promote the expression of a variety of cellular and viral genes including proto-oncogenes [
5,
6].
Intriguingly, an additional open reading frame (ORF) with 56 nucleotide triplets upstream of the starting code of the X gene has been identified [
7‐
10]. Our previous studies have displayed the novel ORF has transcriptional activity [
11] and can be translated with the X gene in frame to form the HBV whole-X gene (wX) [
12]. The HBV whole-X gene, which consists of the novel ORF and X gene and runs as long as 630 bp, codes the HBV whole-X protein composed of 210 amino acids. Studies have shown that the whole-X gene is relatively well conserved in numerous HBV genomes except for the start codon A nt 1205C/T or C/A nt 1330 T replacement mutation resulting in disability of coding whole-X gene. The whole-X gene is identified mainly in HCC patients in Asia or of Asian origin and is presented specifically in HBV C/adr genotype [
13]. According to our knowledge, HBV C/adr genotype is more closely associated with HCC than any other genotypes. Loncarevic et al. reported that all the five HBV DNA clones derived from HCC patients had an intact whole-X ORF. Yang et al. demonstrated the interaction between HBwx and hepatoproteins by using a yeast two-hybrid assay, suggesting it may play a role in the development of carcinoma via modulating signal transduction of protein–protein binding in the liver cells [
14]. These results implicate that HBwx, with extra 56 amino acids longer than HBx, may be related to HCC pathogenesis.
Whereas, the specific function of HBwx in HCC need further investigation. In this study, we examined the expression of HBwx in the liver tissues from patients with HBV-related HCC and investigated its influences on cell growth, cycle, apoptosis and oncological characters in comparison with HBx. We intended to reveal the functional differences between HBwx and HBx and to provide new insight into the possible role of HBwx in HCC pathogenesis.
Methods
Liver tissue specimens
Liver tumor specimens were obtained from 50 patients with HBV-related HCC, and the control liver tissues specimens were from 10 non-HCC patients with HBV infection after hepatectomy. The clinicopathological data of HCC specimens are presented in Table
1. The tissues were collected at the First Affiliated Hospital of Medical College, Xi’an Jiaotong University (Xi’an, China) from January 2002 to June 2009. The study protocols were approved by the Ethical Review Committee of the First Affiliated Hospital of Medical College, Xi’an Jiaotong University, and informed consent was signed by all participants. The methods were carried out in accordance with the approved guidelines.
Table 1
Correlation between HBwx expression and clinicopathologic features in 50 HCCs
Age (y) | | | 0.796 |
≥55 | 13 | 9 (69 %) | |
<55 | 37 | 27 (73 %) | |
Gender | | | 0.756 |
Male | 44 | 32 (73 %) | |
Female | 6 | 4 (67 %) | |
Histological grade | | | 0.095 |
I + II | 35 | 25 (71 %) | |
III | 15 | 7 (47 %) | |
Tumor diameter (cm) | | | 0.063 |
<5 | 22 | 18 (82 %) | |
≥5 | 28 | 16 (57 %) | |
AFP (ng/mL) | | | 0.658 |
<400 | 29 | 19 (66 %) | |
≥400 | 21 | 15 (71 %) | |
Preparation of HBwx polyclonal antibodies
Rabbit anti-HBwx antibodies were produced by commercial Company (Beijing Biosynthesis Biotech, Beijing, China). Peptides (12–14 amino acids) were designed specifically for the conserved regions of HBV pre-X (HBV DNA nt1207-nt1374) by protein sequencing and epitope analysis for antibody production. The peptides of two designed sequences (1#HAWNLCGSSADP, 2#YCGTPSSLFCSQPV) were synthesized and conjugated with KLH protein as the antigen. Immunized rabbit antiserums were collected and purified with antigen specific affinity purification, and then titered by Enzyme Linked Immunosorbent Assay (ELISA).
Immunohistochemical staining (IHC)
Paraffin-embedded liver tissues were cut into 5 μm sections and placed on polylysine-coated glass slides. Antigen retrieval was achieved by pressure cooking for 2 min in citrate buffer (pH6.0). A rabbit anti-human HBwx polyclonal antibody at 1:1280 dilution and a mouse anti-HBx monoclonal antibody (ab235) (Abcam, Cambridge, MA) at 1:500 dilution were used as primary antibodies. Peroxidase-Conjugated AffiniPure Goat Anti-Rabbit IgG (ZB-5301) and Anti-Mouse IgG (ZB-5305) (Zhongshan Goldenbridge Biotech, Beijing, China) were used as the secondary antibodies. The substrate 3, 3′-diaminobenzidine tetrahydrochloride (DAB) was followed by counterstaining with hematoxylin. The negative staining control was performed with cold phosphate buffer solution (PBS) instead of the primary antibody. Immunostaining intensity of HBwx was divided into strong positive (++), scattered positive (+), seldom (±) and negative (−) according to the distribution of positive staining cells in the tissues by 2 independent observers.
Plasmids
The full-length HBV whole-X, X genes were cloned from the plasma of the patients with chronic HBV infection, and subcloned into pcDNA3.1(−), pCMV-Tag2A and pEGFP-C1 vectors respectively. Recombinant plasmids pCMV-Tag2A-wX, pEGFP-C1-wX, pCMV-Tag2A-X and pEGFP-C1-X were further confirmed by DNA sequencing.
Cell culture and transfection
Hepatoma cell lines SK-Hep-1 and SMMC-7721 cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, USA) supplemented with 10 % fetal bovine serum (FBS) (Gibco). HL-7702, a normal liver cell line (Shanghai Institute of Biochemistry & Cell Biology, Shanghai, China), was cultured in the RPMI-1640 medium supplemented 10 % FBS. Transfection was performed by using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to manufacturer’s instruction. Cell lines stably overexpressing HBwx or HBx were established in SK-Hep-1cells by G418 (800/400 μg/ml) selection.
18 Balb/c male nude-mice, 4–6 weeks old, were randomly divided into three groups, and then subcutaneously inoculated with 2 × 106 transformed SK-Hep-1 cells containing pCMV-Tag2A-wX, pCMV-Tag2A-X and pCMV-Tag2A as a control. General state and tumor formation of mice were observed and recorded. The study protocol was approved by the Animal Research Committee of the Medical College of Xi’an Jiaotong University.
The stably transfected SK-Hep-1 cells with overexpressing HBwx or HBx were seeded in 60 mm plates at a density of 500, 1000 or 2000 cells per plate. After 10 days incubation with DMEM medium containing 10 % FBS, the cells were fixed with 4 % paraformaldehyde for 30 min, stained with 0.1 % crystal violet for 20 min, and photographed. Three fixed-size areas were randomly chosen to count the colonies and the averages of colonies were calculated for different cell densities and cell lines.
Cell migration and invasion assay
Cell migration and invasion assay for each overexpressing transformed cell line was performed by using 24-well Millicell (Millpore, Billerica, USA) coated without or with Matrigel (BD Biosciences, New Jersey, USA). 200 μl of 1 × 105/ml cells were transferred onto the transwell chambers and cultured for 24 h allowing the cells to move through the extracellular matrix to the lower chamber. The cells on the underside of the inserts were fixed with 4 % paraformaldehyde for 30 min and stained with 0.1 % crystal violet. Each experiment was repeated at least three times independently. Five randomly selected fields on the fixed transwell chambers were counted with three repeats and photographed. Stained membranes were also discolored in 33 % HAc and absorbance of the elution solutions were measured in 96-well plates with a microplate reader (STAT FAX 2100, USA).
Intracellular localization of HBwx
After being transiently transfected with Green Fluorescent Protein (GFP)-labeled recombinant pEGFP-C1-wX, pEGFP-C1-X and control plasmids, HL7702 cells were observed by fluorescence microscopy (microscope model Nikon Ti-s DS-Ril, Tokyo, Japan) at 48 h after transfection. SMMC-7721 with pCMV-Tag2A-wX, pCMV-Tag2A-X were fixed with 4 % paraformaldehyde for 10 min, permeabilized with 0.3 % Trition X-100 for 10 min, then incubated with anti-FLAG® M2 primary antibodies (F3165) (Sigma, St. Louis, USA) and FITC/TRITC fluorochrome-labeled secondary antibodies (ZF-0312/ZF-0313) (Zhongshan Goldenbridge Biotech). Cell nuclei were stained with 1 μg/ml DAPI for 3 min and then observed by fluorescence microscopy.
Cell proliferation
The effects of HBwx and HBx on cell proliferation were evaluated by the Cell Counting Kit-8 assay (CCK-8) (Dojindo Laboratories, Kumamoto, Japan). 100 μl/well (5 × 103) cell suspensions were inoculated in 96-well, incubated at 37 °C with 5 % CO2 for 24 h, 48 h, 72 h and 96 h. Then, the absorbance was measured at 450 nm using a microplate reader and cell proliferation rate relative to the control was calculated.
Cell cycle and apoptosis
Cell cycle and apoptosis were observed using SK-Hep-1 cell lines stably transfected with pCMV-Tag2A-wX, pCMV-Tag2A-X and control plasmids by flow cytometry analysis (FCM). Cells were harvested (1-2 × 106cells/ml) in logarithmic growth phase, washed with PBS and fixed in 70 % ethanol at 4 °C for at least 1 h. Then, the cells were washed in cold PBS, stained with 50 mg/ml propidium iodide (PI) in the darkness for 30 min and resuspended in PBS at 4 °C before being analyzed by CyFlow® SL (PARTEC Company, Germany). Annexin V-FITC/PI Apoptosis Detection kit (KeyGEN, Nanjing, China) was used to detect cell apoptosis rate induced by 0.1–2.0 μg/ml Adriamycin (ADM) and 0.5–2.5 μg/ml Lipopolysaccharides (LPS) at different concentrations and times (3 h, 6 h, 12 h and 24 h). FCM data were analyzed by using Muticycle AV and FloMax software supplied by the instrument.
Western blot
Stably transfected cells were collected, and cell proteins were extracted by using whole cell extraction buffer (WCEB). Equal amounts of protein were separated in sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) gel and transferred to Polyvinylidene Fluoride (PVDF) membranes (Millipore, Germany), then immunodetection was performed using monoclonal anti-Flag® M2 (F3165, Sigma), monoclonal anti-RKIP/PBP (ab76582, Abcam), polyclonal anti-p44/42 MAPK (Erk1/2) (#9102) and anti-Phospho-p44/42 MAPK (Erk1/2) (#9101) (Cell Signaling, Beverly, USA) antibodies.
Statistical analysis
All statistical analyses were performed by using SPSS 13.0 software (SPSS Inc, Chicago, USA). Descriptive data were assessed using the Pearson’s chi-square test. The data from the experiments were shown as the mean ± SD. Analysis of Variance (ANOVA) was applied to determine the comparability of groups, P < 0.05 being considered statistical significance.
Discussion
HBV is the smallest human hepatotropic DNA virus, which mainly infects host hepatocytes and causes a spectrum of pathological processes from acute hepatitis, chronic hepatitis to serious end-stage liver diseases such as hepatic cirrhosis and primary hepatocellular carcinoma [
16]. Studies of epidemiology and natural history have provided a line of evidence that approximately 25 % chronic HBV-infected individuals will develop HCC [
2,
17,
18]. Although the pathogenesis of HBV-related HCC is still elusive, HBx has been well documented as a risk factor and strongly implicated in hepatocarcinogenesis. HBx contributes to HBV replication and interacts with host factors to be involved in gene expression, cell proliferation, cell cycle progress, apoptosis, DNA repair and genetic stability [
6]. Most importantly, HBx functions as a broad-acting transcriptional transactivator, promoting the expression of a variety of proto-oncogenes including c-myc, c-fos, and c-jun etc. [
19].
Since the identification of the HBwx gene [
7‐
12], previous studies including ours, have shown that HBwx may be associated with HCC [
8,
10]; whereas, its physiological role is largely unknown. In this study, HBwx was expressed in about 72 % liver tumor tissues of patients with HBV-related HCC. Interestingly, small tumor masses contain more HBwx positive cells. In contrast, a significantly smaller number of HBwx positive cells were found in large tumor masses and in tumor with higher atypia. Additionally, HBwx displayed a promoting effect on tumorigenesis and growth in vivo and in vitro as well as cell migration and invasion. Consistent with our published data [
15,
20], those results provided substantial evidence for the involvement of HBwx in hepatocarcinogenesis.
However, the subcellular location of HBwx and HBx in the liver cells is distinct from IHC with tumor tissue and subcellular localization analysis. It is generally accepted that the molecular function of a protein is highly dependent on its subcellular localization. By proteomics and bioinformatics analyses, our previous studies have proved that HBwx functions in carcinogenesis in a way that is different from that of HBx [
20]. To discover the differential biological role of HBwx and HBx, the influence of HBwx on cellular biologic behaviors had been evaluated. In comparison with HBx,
HBwx displayed a higher cell proliferation rate, higher percentage of cells at the S phase, more sensitive to cell apoptosis stimulants. The observation implicates that HBwx may be a weaker tumorigenicity-promoting factor compared with HBx. However, there is no significant correlation between HBwx expression and clinicopathological characteristics of HCC. The differences between HBwx and HBx in hepatocarcinogenesis need thorough mechanism research and a large number of clinical samples to clarify. The present results provide significant information for understanding the mechanism of hepatocarcinogenesis and demonstrate clinical implications that may influence diagnostic decisions and treatment strategies in individual patient.
The differential expression of proteins in the cells with HBwx was investigated by proteomic analyses [
15]. Our data suggest that the HBV whole-X may promote the development of liver cancer through downregulating the expression of RKIP, an inhibitor of the Ras-Raf-1-MEK1/2-ERK1/2 pathway. RKIP expression has been reported to be lost or reduced in prostate cancer, breast cancer, liver cancer and other malignant tumors because its role in inhibiting the RAS/RAF/MEK/ERK signaling pathway [
21], which is activated in over 90 % of HCCs. A substantial evidence has manifested the activation of HBx in hepatocarcinogenesis [
22,
23]. In our observation, the expression of RKIP was inhibited both in cells expressing HBwx and HBx, with increasing p-ERK, which implicates the involvement of RKIP-p-ERK pathway in HBwx carcinogenesis. Nevertheless, the explicit biological role and the specific molecular mechanism of HBwx, such as the effects of proliferation, metastasis and angiogenesis induced by HBwx remain to be studied, further investigation on the detail biological function is carrying out.
Acknowledgments
This work was supported by the Major National Science and Technology Projects for Infectious Diseases (12th Five Year, China) (2012ZX10002007), the National Natural Scientific Foundation of China (No.30671862, No.81572435) and the Foundation of Shaanxi Provincial Science and Technology Plan Projects (2010 K14-01).