Mechanisms Of HBx Deletion Mutant On Human Hepatocyte Transformation And HBx Protein Induces Proliferation And Up-regulates TGF-β1 And CTGF Of Human Hepatic Stellate Cell

Part 1 Mechanisms of HBx deletion mutant on human hepatocyte transformationObjective:Chronic hepatitis B infection and the integration of its X gene(HBx) are closely associated with the development of hepatocelluar carcinoma(HCC).The integrated HBx frequently is truncated or contains point mutations.Some studies indicated HBx mutants have different biological functions compared with wild type HBx and they may play a critical role in HBV-related HCC.Our previous studies also showed HBx deletion mutant can apparently promote the proliferation and malignant transformation of hepatocyte.But its molecular mechanism remains unclear.Based on the advantage of proteomics in carcinogenesis studies, we turned to this new technology -"proteomics",which would help to comprehensively clarify the carcinogenesis of HBx deletion mutant in protein level and provide evidence for HBV-related HCC diagnosis and treatment.MicroRNAs(miRNAs) are small RNA molecules that regulate gene expression post-transcriptionally.MiRNAs play a key role in diverse biological processes,including development,cell proliferation, differentiation,and apoptosis.Accordingly,altered miRNA expression is likely to contribute to human disease,including cancer.In this study,we will investigate the differentially expressed miRNA between human hepatocycte transfected with wild type HBx gene and deletion mutant of HBx gene by microarray,which will be useful for further studies on function of miRNA in the pathogenesis of HBV-related HCC.Methods:(1) In previous studies,we have established QSG7701 cell lines stablely transfected with HBx deletion mutant and wild type HBx gene(namely QSG7701-HBx-d382 and QSG7701-HBx-2215 cells). In this study,HBx mRNA and protein expression in the two group cells were verified by RT-PCR and Western blot respectively.(2) To analyze differentially expressed proteins between QSG7701-HBx-d382 and QSG7701-HBx-2215 cells by proteomics technology:Firstly the total proteins of QSG7701-HBx-d382 and QSG7701-HBx -2215 cells were separated with immobilized PH gradient-based two- dimensional gel electrophoresis(2-DE),After Coomassie staining,gel images were acquired by Image-scanner and then analyzed with the PDQuest software. Two-DE maps of QSG7701-HBx-d382 and QSG7701-HBx-2215 were established.Secondly partial differentially expressed protein spots were incised from gels and digested by trypsin in-gel.Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) was used to analyse partial differentially expressed proteins pots and get peptide mass fingerprinting.Finally SwissPort database searching by Mascot software were used for protein identification.(3) To identify the differentially expressed miRNAs between QSG7701-HBx-d382 and QSG7701-HBx-2215 cells by microarray:Firstly total RNA of QSG7701-HBx-d382 cells and QSG7701-HBx-2215 cells was isolated by Trizol. The concentration and purity of RNA was determined by NanoDrop ND-100,the quality of RNA was assessed by denaturing agrose gel electrophoresis.Secondly labeled with Hy3^TM,the miRNA was hybridized on miRNA array.After hybridization miRNA assay was scanned by GenePix 4000B,image information was transformed into numeral date,then the original date was analyzed using GenePix software.Results:(1) RT-PCR and Western blot results confirmed the mRNA and protein expression of HBx in QSG7701-HBx-d382 and QSG7701-HBx-2215 cells.(2)Well-resolved reproducible 2-DE patterns of two group cells were obtained.A total of 920±24 and 950±27 spots were detected in QSG7701-HBx-d382 and QSG7701-HBx-2215 cells respectively,and the average matching rates were 87.2%and 89.1% respectively.Fifty-five differentially expressed protein spots were detected,among which 32 were up-regulated,23 were down-regulated. Fifteen differentially expressed protein spots got further study.Peptide mass fingerprints(PMF) by MALDI-TOF-MS analysis ennabled the identification of 12 proteins,among which some are involved in cell proliferation,apoptosis and signal transduction.(3) The quality of RNA was high,which suited array hybrization.MicroRNA array hybrization showed that expression of miR-7a,miR-15a,miR-122,miR-17,miR-585,miR-886-5p,miR-886-3p,miR-494,miR-668 was down-regulated in QSG7701-HBx-d382 cells by more than two folds, while expression of miR-443,miR-663,miR-429 was up-regulated by more than two folds.Conclusion:(1)In this study,well-resolved reproducible 2-DE patterns of QSG7701-HBx-d382 and QSG7701-HBx-2215 cells were established,and 12 differentially expressed proteins were characterized, among which some are involved in cell proliferation,apoptosis and signal transduction.These results provide fundamental information for molecular mechanism of HBx deletion mutant induced HCC.(2) miR-7a,miR-15a,miR-122,miR-17,miR-585,miR-886-5p,miR-886-3p,miR-494,miR-668,miR-443,miR-663,miR-429 were differentially expressed between QSG7701-HBx-d382 and QSG7701-HBx-2215 cells. These results indicated these miRNA may be associated with HBx deletion mutant-induced malignant transformation of liver cells,which may play important role in pathogenesis and development of HBV-related HCC. Part 2 Hepatitis B Virus X Protein Induces Proliferation and Up-regulates TGF-β1 and CTGF of Human Hepatic Stellate CellObjective:Although chronic HBV infection is a major cause of liver fibrosis,moreover chronic hepatitis B finally progresses into liver cirrhosis via fibrogenesis at high rates,the mechanism of HBV-induced liver fibrosis has not been elucidated yet.The prevailing hypothesis is that HBV infects hepatocytes that release profibrogenic substances (cytokines,ROS) to activate neighboring HSCs,the main matrix -producing cells in the process of liver fibrosis.In this study,we investigate the hypothesis:whether HBx,a multifunctional HBV protein, can directly interact with HSCs and exert profibrogenic action.Mehthods:We established an in vitro co-culture system with LX-2 cells and a stable QSG7701-HBx cell line which had been transfected with HBx gene.³H-TdR incorporation and flow cytometry were used to determined the effects of HBx on the proliferation of LX-2 cells.The mRNA expression levels ofα-smooth muscle actin(α-SMA),transforming growth factor -β1(TGF-β1),connective tissue growth factor(CTGF) and Colâ… in LX-2 cells were analyzed by semiquantification RT-PCR.The protein expression levels ofα-SMA,TGF-β1 and CTGF were measured by Western blot.In addition,the expression levels of collagen typeâ… (Colâ… ) from the co-cultured media were quantitated using by ELISA.Results:(1) ³H-TdR incorporation in LX-2 cells co-cultured with QSG7701-HBx cells was higher than that with QSG7701-pcDNA3 and QSG7001 cells(31252±2210cpm/well vs 14055±1021cpm/well and 13500±942cpm/well,P＜0.05).(2) the S phase ratio and proliferative index(PI=S+G2/M) of LX-2 cells co-cultured with QSG7701-HBx cells was higher than that of two control groups(P＜0.05).These results suggest HBx can accelerate the progression of G1 to S in cell cycle of LX-2 cells. (3) HBx protein could significantly enhance the mRNA levels ofα-SMA,TGF-β1,CTGF and Colâ… (p＜0.05).(4)HBx protein also could obviously up-regulate the protein expression levels ofα-SMA,TGF-β1 and CTGF(p＜0.05).(5) The expression levels of Colâ… in LX-2 cells co-cultured with QSG7701-HBx cells were about 150%higher than in those with QSG7701-pcDNA3 and QSG7701 cells(500.5±43.4ng/ml vs 200.2±21.6ng/ml and 191.2±20.8ng/ml).These results suggest that HBx enhances the fibrogenesis-related proteins leading to fibrosis.Conclusion:(1) HBx protein can promote the proliferation up-regulate TGF-β1 and CTGF of HSC.it also can promote the synthesis and secretion of colâ… in HSC.This may be a new way of liver fibrogenesis in patients with chronic HBV infection...