The Effect Of HBx On Proliferation And EMT Of Mouse Hepatic Progenitorc Ells HP14.5

AIMEstablish HP14.5cell line which stable expressing Hepatitis B virus X(HBx) and research the effect of expression of HBx on cell proliferation，cell cycle，and epithelial-mesenchymal transition（EMT） the wnt/β-catenin signaling pathway of hepatic progenitor cells，and discuss themechanism of HBV-associated hepatocellular carcinoma(HCC).METHODSThe plasmids of pSEB-Flag-HBx and the packaging plasmids pAmphowere co-transfected into293T cells to make retrovirus，and then infectedmouse hepatic progenitor cells HP14.5， obtained blasticid-resistant clonesof HBx cells(HP4.5/HBx) as expreimental group and paralleled with thevector control HP14.5/Rv(HP14.5/pSEEB-Flag) and HP14.5cell ofnegative control. The expression of HBx were identified by RT-PCR andwestern bIotting. Observed the morphological changes of the cells，the cellproliferation was tested by MTS assay，the cell cycle was measured by flowcytometry， the expression leveIs of Cyclin D1and c-myc m RNA weretested by Real time PCR， the expression leveIs of HBx、Cyclin D1、 c-myc、GSK3β、p-GSK3β（ser-9）、β-catenin Proteins were examined bywestern bIotting and the expression changes of epithelial and mesenchymalmarkers were investigated by RealTime-PCR and Western blotting,ndthemetastatic capacity of HBx infected with HPCs was evaluated throughwound healing assay．RESULTSThe expression of HBx mRNA and protien was confirmed inHP4.5/HBx cell line. Compared with control group,1.The proliferation ofHP4.5/HBx cells were increased significantly (P<0.05)， the mRNAexpressions level of Cyclin D1and c-myc were increased significantly(P<0.05). G1cell proportion reduced while S and G2cell proportionincresedobviously(P<0.05).The protein expression level of Cyclin D1、c-myc、β-catenin and p-GSK3β（ser-9）were increased significantly (P<0.05)except GSK-β.2. HPCs which stable expressing HBx suffered morphologychange from a epithelial charcter to a spindle-like shape．In addition, Theexpressions of N-cadherin、Snail and Vimentin protein and mRNA weresignificantly increased while the expressions of E-cadherin and CK18weredecreased in the HP14.5/HBx cells．Furthermore, the cellular migrationassay shows that transfection of HBx drastically enhanced the migratorypotential of HP14.5cells.CONCLUSIONS1. The hepatic stemcell line stable expressing HBx was constructed successfully.2. Hbx can promote the proliferation and malignant transformation ofhepatic stem cells by activation of Wnt/β-catenin signal pathway.3. HBx can promot EMT and enhance the migratory potential ofHP14.5cells.