乙肝病毒X蛋白上调肝癌细胞缺氧诱导因子-1α的作用和机制研究  

The role of hepatitis B virus X protein in regulation of hypoxia inducible factor-1α and the underlying mechanisms in hepatocellular carcinoma

作　　者：刘利平[1,2] 杨盛力 何婉[1,4] 孙枫林[1,2] 鲍世韵 

机构地区：[1]暨南大学第二临床医学院 [2]深圳市人民医院肝胆外科,广东深圳518020 [3]华中科技大学同济医学院附属梨园医院普通外科,湖北武汉430077 [4]深圳市人民医院肿瘤科,广东深圳518020

出　　处：《中国癌症杂志》2015年第5期333-338,共6页China Oncology

基　　金：国家自然科学青年基金(81402041)

摘　　要：背景与目的:乙肝病毒X蛋白(hepatitis B virus X protein,HBx)和缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)在肝癌发生、发展过程中起重要作用。有研究显示,两者在肝癌组织中的表达呈正相关,但相关机制尚不明确。本研究拟进一步在细胞水平上探讨HBx对HIF-1α的调控作用及机制。方法:用LipofectemineTM 2000包裹HBx表达质粒转染到肝癌Huh7细胞。蛋白[质]印迹法(Western blot)检测Huh7细胞中HIF-1α和HIF-1β蛋白的表达;特异性试剂盒检测HIF-1α转录活性;实时定量PCR(quantitative real-time PCR,q RT-PCR)检测HIF-1α及其靶基因血管内皮生长因子(vascular endothelial growth factor,VEGF)和多药耐药基因1(multi-drug resistance gene 1,MDR1)m RNA表达变化;免疫共沉淀法检测HIF-1α、HBx和希佩尔林道病肿瘤抑制蛋白(protein von Hippel-Lindau,p VHL)间的相互作用。结果:转染HBx质粒后,Huh7细胞中HIF-1α蛋白表达、转录活性及其靶基因VEGF和MDR1的m RNA表达水平明显上调(P<0.05),然而HBx对HIF-1αm RNA表达水平没有明显影响(P>0.05)。同时,HBx显著削弱p VHL介导的泛素化水解蛋白酶降解HIF-1α的活性。免疫共沉淀法检测进一步提示,HBx可直接结合p VHL,而对HIF-1α没有结合作用。结论:HBx可能通过直接结合p VHL,抑制其与HIF-1α的相互作用,从而增强HIF-1α蛋白的稳定性及转录活性。Background and purpose：Hepatitis B virus X protein （HBx） and hypoxia inducible factor-1α（HIF-1α） play key roles in hepatocarcinogenesis and the development of hepatocellular carcinoma. Positive correlation on the expression of these 2 proteins in hepatocellular carcinoma tissues has been found, whereas the underlying mechanisms have not been fully elucidated. This study focused on the role of HBx in regulating HIF-1α and the underlying mechanisms in hepatocellular carcinoma cells. Methods：The expression plasmids were transfected into Huh7 cells with LipofectemineTM 2000. Western blot analysis was applied to detect the expressions of HIF-1αand HIF-1β protein. The transcriptional activity of HIF-1α was detected by the commercial analysis kits. The mRNA levels of HIF-1αand its target genes, including vascular endothelial growth factor （VEGF） and multi-drug resistance gene 1 （MDR1）, were detected by quantitative real-time PCR （qRT-PCR）. Immunoprecipitation analysis was applied to detect the interaction of HIF-1α, HBx and protein von Hippel-Lindau （pVHL）. Results：Huh7 cells transfected with HBx plasmid led to sharp increase of HIF-1αprotein and transcriptional activity, as well as the mRNA of VEGF and MDR1 （P〈0.05）. However, the mRNA level of HIF-1αwas not obviously changed after HBx transfection （P〉0.05）. Meanwhile, HBx also signiifcantly impaired the function of pVHL in mediating the degradation of HIF-1αby ubiquitin hydrolase. This finding was further confirmed by the immunoprecipitation analysis, which showed that HBx could directly bind to pVHL, but not to HIF-1α. Conclusion：HBx may inhibit the inter-activation between pVHL and HIF-1αthrough directly binding to pVHL, and thus enhance the stability and transcriptional activity of HIF-1α.

关 键 词：肝细胞癌 缺氧诱导因子-1Α 乙肝病毒X蛋白 希佩尔林道病肿瘤抑制蛋白 

分 类 号：R735.7[医药卫生—肿瘤]