HBx And HBs Upregulated Expression Of RhoC By Transcription Factor Ets-1

ObjectiveTo study the molecular mechanism of HBV induced RhoC expression,reveal a new mechanism of HBV induced HCC metastasis, and provide anew perspective for the treatment of HCC.Methods1. Bioinformatic methods were used to find the transcription factorsbinding to RhoC promoter region.2. Luciferase report construct of RhoC promoter, expression andinterference plasmid of transcription factors, were generated. SMMC-7721cells were cotransfected with pRL-TK plasmid, RhoC promoter reportconstruct, and expression plasmid of transcription factor or interferenceplasmid of transcription factor. Relative activity of RhoC promoter wasdetected by dual-luciferase report assay system. Total RNA and proteinwere isolated, then qRT-PCR and Western blot were performed to analysethe differential expression of the transcription factor and RhoC.3. RT-PCR and Western blot were performed to detect the differentialexpression between HepG2and HepG2.2.15cells. Over-expressionadenovirus of HBV was generaed and verified. HepG2cells were infected by Ad-HBV, then RT-PCR, qRT-PCR and Western blot were performed toanalyse the differential expression of the transcription factor.4. Expression plasmids of four HBV proteins were generated andtransfected into SMMC-7721cells seperatetly. RT-PCR and qRT-PCR werecarried out to analyze the differential expression of the transcription factorand find out the key protein(s). Interference plasmid(s) of key protein(s)was/were generated and transfected into HepG2.2.15cells, then RT-PCR,qRT-PCR and Western blot were performed to analyze the differentialexpression of the transcription factor and RhoC.Results1. Transcription factor Ets-1, NF-κΒ, Sp1, AP-2α, YY1etc, havemultiple putative binding sites in RhoC promoter fragment were found withsoftwares analysis.2. RhoC promoter luciferase report plasmid, expression andinterference plasmid of transcription factor, were successfully constructed.Over-expression of Ets-1resulted in significant up-regulation of RhoCpromoter activity, mRNA and protein levels. No statistical differences werefound in cells, which were transfected with over-expression or interferenceplasmid of other transcription factors, compared to control groups. Therefor,the transcription factor Ets-1was selected to do further research.3. The expression of Ets-1were remarkably higher in HepG2.2.15cells than that in HepG2cells. The mRNA and protein expression of Ets-1 were higher in HepG2cells infected by Ad-HBV, than that in controlHepG2cells.4. Among four HBV proteins, over-expression of X protein and Sprotein in SMMC-7721cells resulted in up-regulation of Ets-1.Furthermore, interference expression of X protein and S protein inHepG2.2.15cells resulted in significant down-regulation of Ets-1andRhoC.ConclusionThe RhoC expression could be up-regulated by transcription factorEts-1. HBx and HBs could increase expression of Ets-1, whichconsequently contributed to up-regulation of RhoC. These findings mightshow a novel insight of HBV induced HCC and provide new therapeutictargets.