中国癌症杂志
中國癌癥雜誌
중국암증잡지
CHINA ONCOLOGY
2015年
5期
333-338
,共6页
刘利平%杨盛力%何婉%孙枫林%鲍世韵
劉利平%楊盛力%何婉%孫楓林%鮑世韻
류리평%양성력%하완%손풍림%포세운
肝细胞癌%缺氧诱导因子-lα%乙肝病毒X蛋白%希佩尔林道病肿瘤抑制蛋白
肝細胞癌%缺氧誘導因子-lα%乙肝病毒X蛋白%希珮爾林道病腫瘤抑製蛋白
간세포암%결양유도인자-lα%을간병독X단백%희패이림도병종류억제단백
Hepatocellular carcinoma%Hypoxia inducible factor-lα%Hepatitis B virus X protein%Protein von Hippel-Lindau
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，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。
揹景與目的：乙肝病毒X蛋白(hepatitis B virus X protein，HBx)和缺氧誘導因子-1α(hypoxia inducible factor-1α，HIF-1α)在肝癌髮生、髮展過程中起重要作用。有研究顯示，兩者在肝癌組織中的錶達呈正相關，但相關機製尚不明確。本研究擬進一步在細胞水平上探討HBx對HIF-1α的調控作用及機製。方法：用LipofectemineTM 2000包裹HBx錶達質粒轉染到肝癌Huh7細胞。蛋白[質]印跡法(Western blot)檢測Huh7細胞中HIF-1α和HIF-1β蛋白的錶達；特異性試劑盒檢測HIF-1α轉錄活性；實時定量PCR(quantitative real-time PCR，qRT-PCR)檢測HIF-1α及其靶基因血管內皮生長因子(vascular endothelial growth factor，VEGF)和多藥耐藥基因1(multi-drug resistance gene 1，MDR1)mRNA錶達變化；免疫共沉澱法檢測HIF-1α、HBx和希珮爾林道病腫瘤抑製蛋白(protein von Hippel-Lindau，pVHL)間的相互作用。結果：轉染HBx質粒後，Huh7細胞中HIF-1α蛋白錶達、轉錄活性及其靶基因VEGF和MDR1的mRNA錶達水平明顯上調(P<0.05)，然而HBx對HIF-1α mRNA錶達水平沒有明顯影響(P>0.05)。同時，HBx顯著削弱pVHL介導的汎素化水解蛋白酶降解HIF-1α的活性。免疫共沉澱法檢測進一步提示，HBx可直接結閤pVHL，而對HIF-1α沒有結閤作用。結論：HBx可能通過直接結閤pVHL，抑製其與HIF-1α的相互作用，從而增彊HIF-1α蛋白的穩定性及轉錄活性。
배경여목적：을간병독X단백(hepatitis B virus X protein，HBx)화결양유도인자-1α(hypoxia inducible factor-1α，HIF-1α)재간암발생、발전과정중기중요작용。유연구현시，량자재간암조직중적표체정정상관，단상관궤제상불명학。본연구의진일보재세포수평상탐토HBx대HIF-1α적조공작용급궤제。방법：용LipofectemineTM 2000포과HBx표체질립전염도간암Huh7세포。단백[질]인적법(Western blot)검측Huh7세포중HIF-1α화HIF-1β단백적표체；특이성시제합검측HIF-1α전록활성；실시정량PCR(quantitative real-time PCR，qRT-PCR)검측HIF-1α급기파기인혈관내피생장인자(vascular endothelial growth factor，VEGF)화다약내약기인1(multi-drug resistance gene 1，MDR1)mRNA표체변화；면역공침정법검측HIF-1α、HBx화희패이림도병종류억제단백(protein von Hippel-Lindau，pVHL)간적상호작용。결과：전염HBx질립후，Huh7세포중HIF-1α단백표체、전록활성급기파기인VEGF화MDR1적mRNA표체수평명현상조(P<0.05)，연이HBx대HIF-1α mRNA표체수평몰유명현영향(P>0.05)。동시，HBx현저삭약pVHL개도적범소화수해단백매강해HIF-1α적활성。면역공침정법검측진일보제시，HBx가직접결합pVHL，이대HIF-1α몰유결합작용。결론：HBx가능통과직접결합pVHL，억제기여HIF-1α적상호작용，종이증강HIF-1α단백적은정성급전록활성。
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.
