中国癌症杂志 ›› 2015, Vol. 25 ›› Issue (5): 333-338.doi: 10.3969/j.issn.1007-3969.2015.05.003

• 论著 • 上一篇    下一篇

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

刘利平1,杨盛力2,何婉3,孙枫林1,鲍世韵1   

  1. 1. 暨南大学第二临床医学院/ 深圳市人民医院肝胆外科,广东 深圳 518020 ;
    2. 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 ;
    3. 暨南大学第二临床医学院/ 深圳市人民医院肿瘤科,广东 深圳 518020
  • 出版日期:2015-05-30 发布日期:2015-08-11
  • 通信作者: 鲍世韵 E-mail:baomi94@163.com
  • 基金资助:
    国家自然科学青年基金(81402041)。

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

LIU Liping1, YANG Shengli2, HE Wan3, SUN Fenglin1, BAO Shiyun1   

  1. 1.Department of Hepatobiliary and Pancreatic Surgery, Shenzhen People’s Hospital, the Second Clinical Medical College of Jinan University, Shenzhen Guangdong 518020, China; 2.Department of General Surgery, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan Hubei 430077, China; 3.Department of Medical Oncology, Shenzhen People’s Hospital, the Second Clinical Medical College of Jinan University, Shenzhen Guangdong 518020, China
  • Published:2015-05-30 Online:2015-08-11
  • Contact: BAO Shiyun E-mail: baomi94@163.com

摘要:

  背景与目的:乙肝病毒X蛋白(hepatitis B v i r u s 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,qRT-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)mRNA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,pVHL)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mRNA表达水平明显上调(P<0.05),然而HBx对HIF-1α mRNA表达水平没有明显影响(P>0.05)。同时,HBx显著削弱pVHL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pVHL,而对HIF-1α没有结合作用。结论:HBx可能通过直接结合pVHL,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。

关键词: 肝细胞癌, 缺氧诱导因子-l&alpha, 乙肝病毒X蛋白, 希佩尔林道病肿瘤抑制蛋白

Abstract:   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-1α and HIF- 1β protein. The transcriptional activity of HIF-1α was detected by the commercial analysis kits. The mRNA levels of HIF-1α and 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-1α protein and transcriptional activity, as well as the mRNA of VEGF and MDR1 (P<0.05). However, the mRNA level of HIF-1α was not obviously changed after HBx transfection (P>0.05). Meanwhile, HBx also significantly impaired the function of pVHL in mediating the degradation of HIF-1α by 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-1α through directly binding to pVHL, and thus enhance the stability and transcriptional activity of HIF-1α.

Key words: Hepatocellular carcinoma, Hypoxia inducible factor-lα, Hepatitis B virus X protein, Protein von Hippel-Lindau