Study On The Effects Of Hepatitis B Virus Trans-regulators On Autotaxin Expression

Objective: Hepatitis B virus(HBV)infection remains one of the main problems of global public health and the pathogenesis of hepatitis B and its complications need to be further research.This study aims to investigate the effects of hepatitis B virus(HBV)trans-regulators,X protein(HBx)as well as pre S2 protein(pre-S2),on autotaxin(ATX)expression and thus provide experimental basis for clearing and defining the pathogenesis of hepatitis B and its complications.Methods: The promoter of ATX and the genes of HBx and pre-S2 were obtained by PCR amplification and then cloned into pGL3-Enhancer and pcDNA3.1(+)to construct a luciferase reporter gene recombinant vector of ATX promoter,the pGL3-ATX,as well as the eukaryotic expression recombinant vectors of the pcDNA3.1(+)-HBx and the pcDNA3.1(+)-pre-S2,respectively.The effects of HBx and pre-S2 on ATX promoter were examined by measuring luciferase activity.Furthermore,we were established the stable cell expressing HBx or pre-S2 gene,HepG2.HBx cell and HepG2.pre-S2 cell.The expression of ATX protein was detected by Western blotting.Results: Both of the recombinant vectors,pcDNA3.1(+)-HBx and pGL3-ATX,were confirmed to be constructed as their designs by double-enzyme digestion and DNA sequencing.The luciferase activity detection showed that the luciferase activity of pGL3-ATX was 10 times as much as that of empty vector pGL3-basic(P < 0.001).The results of cotransfection showed that the luciferase activity of the pcDNA3.1(+)-HBx + p GL3-ATX group and the pcDNA3.1(+)-pre-S2 + pGL3-ATX group were 1.47 times and 1.15 times as much as that of the empty vector pcDNA3.1(+)+ pGL3-ATX group(P < 0.001).HBx and pre-S2 were stably expressed in HepG2 cells,respectively.Western blotting assay showed that the ATX expression was increased in HepG2.2.15 cells,which is a cell line HBV virus stably transfected by HBV virus,and Hep G2.HBx,the cell stably expresses HBx,as well as HepG2.pre-S2,the cell stably expressies pre-S2,compared with Hep G2 cells.Conclusion: 1.The promoter sequences of ATX,which has promoter activity,and HBx gene were cloned;2.The eukaryotic expression recombinant vector of the pcDNA3.1(+)-HBx and the recombinant vector of ATX promoter with a luciferase reporter gene,the pGL3-ATX,were constructed;3.The cells,HepG2.HBx and HepG2.pre-S2,which can stably express HBx and pre-S2,respectively,were constructed.4.Both HBx and pre-S2 can enhance the activity of ATX promoter;5.The amounts of ATX protein expression successively increase in Hep G2,HepG2.pre-S2,HepG2.HBx and HepG2.2.15;6.HBx and pre-S2 can activate ATX promoter and thus up-regulate ATX expression,suggesting that HBV infection can enhance the ATX/lysophosphatidic acid signaling via increasing ATX expression by its trans-regulators,HBx and pre-S2,thereby affecting the physiological and pathological processes of the host cells.