FGA Acts As A Novel HBsAg Binding Protein And The FGA-HBsAg Interaction Affects The Function Of HepG₂Cell

Objective: To confirm the FGA as a binding protein of HBs and toinvestigate the function and involved mechanism between itsinteractions during Hepatocellular carcinoma (HCC) development.Methods:(1) FGA gene was amplified by RT-PCR with the total RNA ofhuman HepG2cell as template.The PCR fragments were cloned intopcDNA6vector to construct recombinant eukaryotic expressionplasmids.Whole S gene recombinant plasmid as a template by PCRamplification of the major S gene, building pCMV4-Flag-HBS expressionvector（HBS was constructed in our lab.）.293T cells weretransfected with the recombinant plasmids.The expressions of FGA andHBS in293FT cells were used to certificate by Western blot.(2) Thebinding and co-localization between HBs and FGA was confirmed usingco-immunoprecipitation and confocal microscopy.(3) Applied stablytransfected vector-based small interfering RNA (siRNA) to knock downthe expression of FGA in HepG2cells which overexpressed the HBsAgprotein(to establish four of stable cell lines (pCMV4+pU6,pCMV4+siFGA, HBs+pU6, and HBs+siFGA)), and employed CCK8assay, flowcytometry and transwell assay to analyze cell proliferation,apoptosis and migration and invasion, respectively. The involvedmechanisms were further investigated using protein array and westernblot analysis.Results:(1) An interesting band about72KD was visible in the resultof Western blot, which was same to the expected results; About26KD in the expected location of the HBS protein. Co-location in thecytoplasm of HepG2cells was showed by confocal microscopy.(2)Endogenous co-immunoprecipitation successfully confirmed that FGAis a binding protein of HBs.(3) Interaction between HBs and FGAsignificantly enhanced the proliferation of HepG2cells. However,their interaction significantly induced the apoptosis and had noinfluce on migration and invasion in HepG2cells.Both of FGA and HBspromoted the proliferation of HepG2cells. FGA showed the suppressiverole on HepG2cell apoptosis and metastasis and invasion. HBspromoted the HepG2cell apoptosis and metastasis and invasion.(3)Moreover, knockdown of the FGA protein decreased the expressionlevels of the prosurvival factors Bcl-XL and Mcl-1, increased theexpression of the proapoptotic proteins, and decreased thephosphorylation levels of Akt in HepG2cells.Conclusion:(1) pcDNA6-FGA-myc had been constructed successfully.(2)FGA is a novel binding protein of HBs.(3) FGA protein effected theproliferation，apoptosis，metastasis and invasion in HepG2cells.(4)The interaction of FGA and HBS can regulate the expression of Bcl-2and Akt cell signaling pathway proteins.