Background
Hepatocellular carcinoma(HCC) development closely associated with infected by hepatitis B virus(HBV). HBV-X protein(HBx), a small regulatory protein of HBV that has been require for contributing to the onset and progression of HBV-related HCC [
1-
3]. However, the molecular mechanisms involved in HBx-mediated hepatocarcinogenesis remain to be fully elucidated. HBx emerged transcriptional activity on a variety of viral and cellular promoters [
4-
6]. HBx does not directly bind to genomic DNA of host cells, but has been shown to interact with components of basal transcription machinery [
7,
8] and several transcription factors, such as p53, HIF-1α and E4F1 [
9-
11]. However, documents evidenced that the localization of HBx predominantly in the cytoplasm, HBx harbors a function to activate signal transduction cascades, including phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT) [
12,
13] and mitogen-activated protein kinase(MAPK) [
14]. Activation of these signal pathways may contribute to HBx-mediated effects on driving malignant transformation of liver cells.
Alpha fetoprotein(AFP) is an early biomarker of HCC diagnosis, promote tumor cells proliferation effects of AFP have been reported by several groups [
15,
16], furthermore, data indicated that AFP play pivotal role in the hepatocarcinogenesis [
17]. Our investigations found that these effects of AFP maybe mediated by AFP receptors(AFPR) [
18,
19], cytoplasmic AFP activated PI3K/AKT signal pathway to promote expression of some oncogenes and proliferation of HCC cells [
16,
20,
21]. HBx priors to induce expression of AFP and AFPR to activate PI3K/AKT signal pathway in normal liver tissues and cell lines [
22]. Because HBx was a critical factor for HBV driving development of HCC, HBx activates Wnt/β-catenin and Src kinase led to malignant transformation of liver cells [
23], and Src plays important role in HCC development [
24]. In this study, we discovered that HBx priors to induce expression of AFP and AFPR in normal liver tissues and liver cell lines via activating PI3K/AKT signal, and AFP promoted expression of Src was mediated by AFPR, AFPR signal possess a character to activate PI3K/AKT. Our results supported that AFP and AFPR as potential stimulated factors for HBx inducing hepatocarcinogenesis.
Methods
Clinical specimens collected
Archived clinical specimens were originally collected during hepatectomy of 71 patients at Hainan Provincial People’s Hospital between October 2008 and September 2014. Of the 71 patients, 49 were male and 22 were female. The ages ranged between 22–76 years with an average age of 49.8 years. All enrolled patients were treated with radical surgery and received no other treatments. HBV infection was diagnosed by a test of serum hepatitis B surface antigen, and circulating AFP plasma level was measured by enzyme-linked immunosorbent assay. Clinical data were obtained by retrospective chart review. Follow-up was available for all patients. A section of liver tissue about 2.0 × 2.0 × 2.0 cm was obtained from each patient immediately after the surgery. About 1.0 × 1.0 × 1.0 cm tissue samples were fixed in 10% formalin, embedded in paraffin, and routinely stained with hematoxylin and eosin. Specimens were assessed blindly and independently by two pathologists. In case of interobserver disagreement, final decisions were achieved by general consensus. All selected patients were diagnosed by histopathologic evaluation. About 1.0 × 1.0 × 1.0 cm tissue specimens were stored in formalin and liquid nitrogen. The study protocol was approved by the Ethical Committee of Hainan Provincial Peoples’ Hospital and the Science Investigation Ethical Committee of Hainan Medical College. Written informed consent was obtained from all participants.
Immunohistochemical stained
All of clinical patients’ liver tissues were performed by immunohistochemical staining. Following deparaffinization and antigen retrieval, the slides were blocked with 3% hydrogen peroxide for 10 minutes and then incubated with mouse anti-AFP, AFP receptor(AFPR), or Src-directed antibodies (Abcam Biotech Company, Cambridge, UK) at 4°C overnight. After washing, sections were incubated with secondary goat anti-mouse antibodies (Merck-Calbiochem) at room temperature for 60 minutes and then developed with 3,3-diaminobenzidine chromogen solution in 3,3-diaminobenzidine buffer substrate(Merck Chemicals). Sections were visualized with 3,3-diaminobenzidine and counterstained with hematoxylin. All sections were visualized by microscope(Olympus).
Cell lines
Human normal liver cell lines, L-02 cell was purchased from the Shanghai Institution of Cellular Biology, Science Academy of China and were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. The AFP-producing and HBV-infected cell line PLC/PRF/5 was gift from the Department of Cell Biology, Peking University Health Science Center and were maintained in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% fetal calf serum. All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO2.
Generation of HBx-expressing constructs and transfects
Construction of the HBx-expressing construct (pcDNA3.1-
HBx) and the primer used for HBx gene amplification have been previously described [
22]. Lipofectamine® 2000(Beyotime Biotechological Corp, Haimen, Jaingsu, China) was used to promote pcDNA3.1-
HBx vectors transfected into L-02 cells. Stably transfected L-02 cells were screened using G418 (Cat No. 30-234-CR, MediatechInc, Manassas, USA) and named L-02-X.
Western blotting analysis
Western blotting was employed to assess the protein levels of AFP, AFPR and Src. Twelve clinical patients’ specimens that were randomly selected for detecting and these protein expressed in cell lines as described previously [
21,
22]. The cells were co-treated Ly294002 or GDC-0941(MedChem) with AFP(Sigma), and the expression of Src, pAKT(Ser473) were detected by Western blotting.
Localization of proteins were observed by laser confocal microscopy
The staining procedure for laser confocal microscopy observing has been previously described [
22]. Briefly, cells were fixed in 4% paraformaldehyde and incubated with mouse anti-human AFPR, AFP and Src antibody for 12 hours. FITC-conjugated or TRITC-conjugated secondary anti-mouse immunoglobulin G was added and incubated for 2 hours, followed by the addition of 100 μL DAPI (1 μg/mL) and 30 minutes of incubation. Cells were visualized with the Leica TCS-NT SP2 laser confocal microscopy (Leica Camera).
Soft agar formation assays were performed to compare the clonogenic potential of L-02 and L-02-X cells in semisolid medium. Briefly, 5000 cells of L-02 or L-02-X were mixed with 0.5% soft agar and plated on a layer of 0.8% of bottom agar in 6-well plates. 2 mL of complete medium was added on the top of agar. Cells were fed twice a week, and the plates were incubated for 14 or 21 days at 37°C with 5% CO2. Colonies were photographed and counted with a Nikon inverted microscope(Nikon Corp., Tokyo, Japan).
Statistical analysis
The results of multiple observations were presented as the mean ± SD of at least three separate experiments. Statistical significance was determined using x2 and the student’s t test (SPSS 11.5 software).
Discussions
HBx proteins play important roles in the development of HBV-related HCC through the activation of growth signal pathways and the inactivation of tumor suppressive pathways, such as transcripted activity of p53 [
9,
27]. In this study, we found that overexpression of AFP, AFPR and Src in cirrhosis and HBV-related HCC tissue samples, and showed that expression of AFPR and AFP prior to expression of Src during the progress of HBV-related hepatocarcinogenesis. These results implied that HBx is involved in AFPR and AFP overexpression during HBV infection. HBx inactivation transcripted activity of p53 to alleviate p53 mediated repression of AFP expression [
9], HBx also stimulated expression of Src via activating PI3K signal pathway [
28], whereas, in this study, we despite found that HBx upregulated expression of AFPR, but the role played of HBx in regulating expression of AFPR is still unclear. Recently, we found that HBx promoted AFPR expression maybe involve in activating PI3K/AKT signal [
22]. These results clued to that activation of PI3K/AKT signal was critical procedure for HBx driving malignant transformation of liver cells.
Recently, documents reported that AFP played pivotal role in HCC development and malignant behavior of liver cells [
29-
31], cytoplasmic AFP activated PI3K/AKT signal to stimulate expression of Ras, CXCR4 and Src through inhibiting activity of PTEN [
16,
21], AFP also inhibited the PI3K/AKT pathway through promoting ubiquitination of PTEN to stimulated malignant phenotype of HCC cells [
32]. HBV infection caused malignant transformation of liver, during this course,
AFP gene was activated in liver cells, so AFP was used as a tumor marker for early warning origination of HCC in clinical diagnosis. Previously, we have found that AFP enhanced proliferation of HCC cells was mediated by AFPR, AFPR was identified as G-protein combined receptor, AFPR signal mediated cAMP and [Ca
2+]i transduction of receptor signal to promoted expression of N-Ras and c-myc [
19], these results implied that AFPR signal was also a critical factor for HCC development. In this investigation, the results indicated that human normal liver cells, L-02 were transfected with HBx-expressed vectors been able to stimulate expression of AFPR priors to the expression of AFP and Src, the results implicated that AFP played important role in inducing malignant transformation of liver cells and enhancing HCC cells malignant behavior was mediated by AFPR.
Activation of Src foreshowed the occurrence of cancer. HBx induced HCC development involve in activation of Src [
33,
34]. In the present study, clinical data displayed that during progression of HCC, elevated expression of Src in liver cells were closely associated with infection of HBV, these results implied that HBx stimulated expression of Src plays an important role in HBV promoting development of HCC. HBx enhanced proliferation and anti-apoptosis and autophagy through activating transduction of PI3K/AKT signal pathway [
13,
35], and PTEN specific suppression of HBx-mediated cell survival through inhibiting PI3K pathway in human normal liver cells, Chang liver [
36]. In this investigation, our results indicated that HBx induced expression of AFPR and location in membrane of L-02-X cells, the expression of AFPR also existed in HBV positive human liver cancer cells PLC/PRF/5. While these cells were treated with AFP, the expression of Src was stimulated, and PI3K specific inhibitor Ly294002 and GDC0941 were capable of withhold the role of AFP. These effects implicated that AFP promoted expression of Src and phosphorylation of AKT(Ser473) in AFPR positive cell lines was mediated by AFPR, it also proved that AFPR possessed a characteristic to activate transduction of PI3K/AKT signal pathway. The present study is the first time that discovered AFPR signal activated PI3K/AKT through signal cross-talk or AFPR maybe plays a role model of tyrosine protein kinase receptor. Recently, we found that HBx driven expression of AFP to activate transduction of PI3K/mTOR signal, stimulated expression of Src and CXCR4 in human normal liver cells [
37], AFP also played role in promoting migration of HCC cells [
38]. In this investigation, soft agar cultured experiment demonstrated that HBx induced expression of Src in normal liver cell line was able to promote colonigenesis and growth in soft agar, implied HBx driven malignant phenotype of liver cells involve in promoting expression of Src. Notwithstanding, our previously study results have confirmed that cytoplasmic AFP promoted proliferation and anti-apoptosis of HCC cells through activating growth signal and inhibiting apoptotic signal [
21,
39,
40], but in this study, we found that HBx induced expression of AFPR and AFP to promote expression of Src in normal liver cells, activation of AFPR signal was a critical factor for HBx driving HCC occurrence. AFPR maybe is applied as a novel bio-target for therapeutics of HCC.
Acknowledgments
This work was supported by the National Natural Science Foundation of China(No. 81360307, 81260306, 81160261, 31060164, 30960153); Key Program of Science and Technology, Ministry of Education of China(No.211146); Key Projects of Science and Technology, Hainan Province(No. ZDXM 20110038); New Century Excellent Talents in China(NCET-10-0124) Natural Science Foundation of Hainan Province(309034, 310044, 811208, 814293) and Fund of Hainan Province Social Development(2015SF03).
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Competing interests
The authors who have taken part in this study declared that they have no competing interests.
Authors’ contributions
Conceived and designed the experiments: ML. Performed the experiments: MZ, JG, WL, HX, YL and XD. Analyzed the data: MZ, YC and XJ. Contributed reagents/materials/analysis tools: SF. Wrote the manuscript: ML and JG. All the authors read and approved the final manuscript.