乙型肝炎病毒X蛋白增强人血管生成因子bFGF基因启动子转录活性  

Molecular mechanisms on HBx stimulating bFGF gene transcription

作　　者：马叶女 朱文琦 李波 侯俊 卞成蓉 刘妍 孙杰 鲍春梅 张浩 李伯安 MA Yenv;ZHU Wenqi;LI Bo;HOU Jun;BIAN Chengrong;LIU Yan;SUN Jie;BAO Chunmei;ZHANG Hao;LI Boan(Treatment and Research Center for Infectious Diseases,the Fifth Medical Center of PLA General Hospital,Beijing 100039,China)

机构地区：[1]解放军总医院第五医学中心感染病医学部研究所,北京100039 [2]解放军总医院第五医学中心感染病医学部检验科,北京100039 [3]河北燕达医学研究院科研事业部,廊坊065201

出　　处：《传染病信息》2023年第6期492-496,502,共6页Infectious Disease Information

基　　金：河北省创新能力提升计划(22567663H)。

摘　　要：目的检测乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)对人血管生成因子bFGF基因转录的激活作用并确定其作用于bFGF基因启动子的区域,分析HBx激活bFGF基因转录可能的分子机制,为揭示HBx促进肝癌发生的分子机制及发现新的治疗靶点。方法运用高保真DNA聚合酶从人肝癌HepG2细胞cDNA文库中扩增出bFGF基因启动子DNA序列;从含完整HBV DNA序列的表达载体中扩增HBx编码序列并克隆入真核表达载体p CDNA3-FLAG中,将HBx表达载体转染人肝癌HepG2细胞中,裂解细胞进行蛋白SDS-PAGE电泳,Western blot检测HBx蛋白的表达;将HBx表达载体和含bFGF基因启动子不同区段的荧光素酶报告载体共转染肝癌HepG2和SMMC-7721细胞,检测HBx对bFGF基因启动子转录活性的影响;运用Western blot检测在肝癌细胞中HBx过表达对ERK磷酸化的影响。结果成功克隆了HBx并使其在肝癌细胞中得到表达;成功克隆了bFGF基因启动子2.2 kb、1.4 kb、720 bp和360 bp的区段;在肝癌细胞SMMC-7721和HepG2细胞中HBx的表达能升高bFGF基因启动子的活性,且对上述4个区段的活性均增加约5倍;HBx以剂量依赖的方式增强bFGF基因启动子的活性;HBx高表达在肝癌HepG2细胞中不能升高ERK1/2的磷酸化水平。结论HBx能够激活bFGF基因启动子的活性,这种激活作用位于HBx启动子转录起始位点上游360 bp片段内,HBx促进bFGF基因转录不依赖于ERK1/2的磷酸化作用,提示HBx可能通过其它信号通路作用于bFGF基因的转录过程。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.

关 键 词：肝癌发生 血管生成因子 HBX BFGF 基因转录 信号转导 

分 类 号：R512.62[医药卫生—内科学]