Interaction Of The Hepatitis B Spliced Protein With TGF?1I1 Contributes To Epithelial-mesenchymal Transition Of Hepatoma Cell Induced By TGF-?1

Human hepatitis B virus(HBV) infection can result in acute and chronic hepatitis, cirrhosis and eventually hepatocellular carcinoma(HCC),andthe variations of the viral genome play an important role in the process of disease. HBSP(Hepatitis B spliced protein) is a protein encoded by 2.2 kb singly spliced variant of hepatitis B virus genome which spliced at the nucleotides position 2447 nt-489 nt.Previous studiesshowed that HBSP correlated with liver cirrhosis andhepatic fibrosis,however, the mechanisms involved are not fully elucidated yet. To investigatethe pathogenic mechanism of HBSP,yeast two-hybrid screeningwas used in our previous study and it was found that HBSP couldinteract withtransforming growth factor-?1-induced transcript 1(TGF-?11I1, also known as Hic-5), which contributed to epithelial mesenchymal transition(EMT). In the present study,we establishedcell linesstably expressing HBSP(named as Huh7-HBSP-flag-HIV cells)and control Huh7-flag-HIV cellsby lentivirus infection. After the additionof 5ng/ml TGF-?1for 24 hours, the morphology changes of Huh7-HBSP-flag-HIVcells were observed. Huh7-HBSP-flag-HIV cells converted from close-knit epithelial shapeinto loose mesenchymal cells. Then the total proteins were extracted from cells and examined by westernblotting using specific antibodies againsta series of EMT markers. We found thatthe expression of epithelial markers were reduced markedly,such as E-cadherin, Claudin-1, ?-catenin. And mesenchymal markerofN-cadherin were upregulated dramatically. Then, TGF-?1I1-si RNA were transfected into cells,and the expression of epithelial and mesenchymal marker proteins were reversed comparing with before. The abilitiesof migration and invasion of cells weredetermined by wound-healing and transwell invasion assay.Huh7-HBSP-flag-HIVpromoted migration and invasion mobility. The study demonstrates that the interaction of Hepatitis B Spliced Protein(HBSP) with TGF-?1I1 may enhance epithelial-mesenchymal transition(EMT) of hepatocellularcarcinoma induced by TGF-?1 and contributes to its migration and invasion mobility.