目的 探讨乙型肝炎病毒Ⅹ蛋白(HBx)抑制细胞色素P450 2E1 (CYP2E1)表达的作用机制及其对肝细胞癌转移的影响.方法 将CYP2E1启动子区域划分为10个不同长度的片段并分别克隆到质粒PGL3-Basic上,与HBx表达载体(pCMV-2B-FLAG-Ⅹ)共转染于HepG2细胞中测定荧光素酶活性以确定HBx作用区域.凝胶电泳迁移率试验分析转录因子与DNA的结合；CCK-8检测细胞的增殖；细胞的侵袭测定采用Martrigel试验.增殖试验、侵袭试验数据比较用t检验；荧光素酶活性数据比较采用方差分析.结果 通过对CYP2E1启动子的缺失分析,重组子P8(删除-483 ～-274bp后的P7部分)活性与P7比较明显增高,达216.69％(F=142.13,P=0.002),确定HBx作用区域位于P7 (-483 ～-274 bp),进而通过MatInspector分析该区域存有肝细胞核因子4α (HNF4α)与胆固醇调节元件结合蛋白-1(SREBP-1)两转录因子结合位点并对该两序列进行突变分析,将mut-HNF4 α、mut-SREBP-1及mut-Duel重组质粒与或不与pCMV-2BFLAG-Ⅹ蛋白表达载体转染HepG2细胞,测定荧光素酶活性,分别为8.6％、24.44％、147.24％,进一步证实转录因子HNF4 α与SREBP-1参与HBx对CYP2E1的调控(F=112.24,P=0.001);凝胶电泳迁移率试验结果显示转录因子HNF4 α与SREBP-1是通过与CYP2E1启动子的结合而发挥作用；CCK-8结果显示转染pCMV-2B-FLAG-Ⅹ蛋白的HepG2细胞的增殖吸光度值A为0.86±0.11,未转染组增殖吸光度值A为0.73±0.09,两组比较,差异有统计学意义(t=5.62,P=0.031); HepG2细胞的侵袭能力也强于对照组,穿膜细胞数为(40.0±8.1)个,显著高于对照组穿膜细胞数(18.0±3.o)个(t=21.54,P=0.001);加入PI3K与JNK信号通路抑制剂Wortmannin、SP600125后可抑制HBx对CYP2E1mRNA与蛋白水平表达的促进作用及对细胞增殖、侵袭的促进作用,提示PI3K与JNK信号通路参与该调控.结论 HBx可能通过PI3K与JNK信号通路激活转录因子HNF4 α与SREBP-1,然后作用于CYP2E1启动子抑制CYP2E1的表达,进而促进肝癌的生长与侵袭.%Objective To explore the mechanism of hepatitis B virus Ⅹ protein (HBx)-mediated inhibition of CYP2E1 expression and its significance in hepatocellular carcinoma (HCC) metastasis.Methods A deletion series and mutagenesis series of human CYP2E1 promoter sequence was co-transfected with the HBx expression vector pCMV-2B-FLAG-Ⅹ into the human HepG2 hepatoma cell line.Reverse transcription-PCR and real-time PCR were used to evaluate the effects of HBx on CYP2E1 promoter activity.The luciferase reporter gene assay was used to identify the HBx-responsive region in the CYP2E1 promoter.Electrophoretic mobility shift assay was used to detect the protein complexes binding to nucleic acids in the CYP2E1 promoter.Martrigel invasion assay was used to examine effects of HBx-inhibited CYP2E1 on invasiveness.Results Analysis of the deletion series and mutagenesis series led to identification of two regions of sequence in the CYP2E1 promoter that are important in HBx-mediated modulation of CYP2E1 activity in HepG2 cells (F =112.24,P =0.001).Both HNF4α and SREBP-1,which directly interact with CYP2E1 promoter sequences,were implicated in the mechanism of HBx-mediated modulation of CYP2E1 promoter activity.In addition,PI3K and JNK pathways were involved in the HBx-mediated modulation (t =8.56,P =0.0012 and t =10.25,P =0.0009 respectively).HBx-mediated repression of constitutive CYP2E1 led to increased invasiveness.Conclusion HBx-mediated inhibition of CYP2E1 expression may promote HCC by increasing tumor progression and invasiveness through modulation of the PI3K and JNK signaling pathways.
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