Angiotensin Ⅱ Promotes Epithelial-mesenchymal Transition Of Hepatocellular Carcinoma Cells Through High Mobility Group Protein B1 Immediated By E4F1

Background and ObjectivesHepatocellular carcinoma has a high prevalence and migrate early,resulting to its poor prognosis.Early studies showed that angiotensin Ⅱ(AngⅡ)can promote tumor metastasis in gastric carcinoma and breast carcinoma,while in the HCC studies were few.High mobility group protein B1(HBGB1)was a hot research area in the recent years in tumor research.This study was aim to evaluate whether Ang Ⅱ can improve hepatocellular carcinoma(HCC)cell line invasion,migration and epithelial mesenchymal transition(EMT).To assess whether high mobility group protein B1(HBGB1)can affect Ang Ⅱ to improve HCC cell line invasion,migration and EMT.At last,screening possible transcript factors of HMGB1 promoter,then make sure with the stimulation of Ang Ⅱ,the specific transcript factor can improve HMGB1 promoter expressionMethods1.HepG2,SMMC7721 and Huh-7 cells were cultured under the stimulation of Ang Ⅱ,the scratch wound healing assay and trans-well assay were used to assess cells’ invasion and migration ability.Western blot was used to assess eptihelial mesenchymal transition(EMT)related proteins ecadherin and vimentin.2.HMGB1 over expression vector and siRNA were constructed to transfer HepG2,SMMC7721 and Huh-7 cells,the scratch wound healing assay,trans-well assay and EMT related proteins assessment were done again to evaluate whether HMGB can influence HCC cells invasion,migration and EMT.3.At last,double gene reporting system was used to assess HMGB 1 promoter activity.Transcript factors were screened then qPCR,WB and ChIP were used to confirm the relationship between specific transcript facor and HMGB 1 promoter.Results1.The scratch wound healing assay andtrans-well assay showed that the invasion and migration ability of HepG2,SMMC7721 and Huh-7 cells were significantly increased under the treatment of Ang Ⅱ(P<0.05).The western blot results suggested that HMGB 1 was significantly up-regulated in hepatocellular carcinoma(HCC)cells(HepG2,Huh-7,SMMC-7721)under the treatment of Ang Ⅱ(p<0.05).2.We successfully constructed the over-expressed HMGB1 vector and HMGB1 siRNA and transfected HCC cells with the stimulation of Ang Ⅱ,the invasion and migration ability of HMGB1 overexpression HCC cells under the treatment of Ang Ⅱ were significantly increased compared negative controls(NC),ecadherin was down-regulated while vimentin was up-regulated;compared with NC,the invasion and migration ability of HMGB1-siRNAHCC cells under the treatment of Ang Ⅱ were not altered,the expression of ecadherin and vimentin were not altered.3.Nineteentranscript factors(TFs)were predicted by the Promoter 2.0 Prediction Server and TFsitescan.The real-time qPCR was used to evaluated the expression levels of these TFs.E4F1 was the only TF abnormally elevated in HCC cells(HepG2,Huh-7,SMMC-7721)treated under the treatment of Ang Ⅱ.WB and ChIP assay showed E4F1 expressed higher than other TFs,under the stimulation of Ang Ⅱ.ConclusionsIn conclusion,E4F1 can be activated by Ang Ⅱ and combined with HMGB1 promoter region to promote HMGB1 expression,then enhance Ang Ⅱ to induce HCC cells’invasion and migration ability and EMT.