Molecular mechanisms on HBx stimulating bFGF gene transcription

MA Yenv; ZHU Wenqi; LI Bo; HOU Jun; BIAN Chengrong; LIU Yan; SUN Jie; BAO Chunmei; ZHANG Hao; LI Boan

doi:10.3969/j.issn.1007-8134.2023.06.002

Abstract：Objective To detect the stimulating effects of HBx on bFGF gene transcription and determine its interaction regions and to define its molecular mechanisms,which will provide new clues for new therapeutic targets and to find patients with high risk to development liver cancer.Methods bFGF promoter was amplified from HepG2 cDNA library with high fidelity DNA polymerase and cloned into pGL4 vector with luciferase reporter and was confirmed by DNA sequencing.HBx coding DNA sequence was amplified and inserted into pCDNA3-FLAG expression construct,which was then transfected into hepatic HepG2 cells and HBx expression was measured by SDS-PAGE and Western blot.The effects of HBx on bFGF promoter activity was determined by cotransfection of HBx expression constructs and various bFGF promoter region-containing luciferase plasmids.HBx overexpression on ERK phosphorylation was detected by Westernblot.Results HBx expression vector was successfully cloned and got expressed in hepatic cells.various bFGF promoter regions namely 2 kb,1.4 kb,720 bp and 360 bp were rightly inserted into luciferase reporter vector.In both SMMC-7721 and HepG2 cells,HBx overexpression enhanced bFGF promoter activity about 5 fold,furthermore,HBx consistently activates all of the 4 promoter regions.Yet HBx overexpression could not change the phosphorylation level of signaling molecule-ERK.Conclusions HBx stimulates bFGF promoter transcription activity and the effects of HBx may lie with 350 bp before transcription start site.HBx action on bFGF promoter does not depend on ERK phosphorylation,suggesting that HBx works through other signaling pathways to regulate bFGF gene transcription.