低氧对人肝癌HepG2细胞中HIF-1α及miR-210表达的影响  

Effects of hypoxia on the expressions of hypoxia-inducible factor-1 alpha and miR-210 in hepatocellular carcinoma HepG2 cells

作　　者：徐凌[1] 王锋[1] 卫巍[1] 戴维奇[1] 何珊珊[1] 王兴鹏[1] 郭传勇[1] 

机构地区：[1]同济大学附属第十人民医院消化内科,上海200072

出　　处：《肿瘤》2011年第6期502-507,共6页Tumor

基　　金：国家自然科学基金资助项目(编号:81072005)

摘　　要：目的:研究低氧对人肝癌细胞株HepG2细胞表达缺氧诱导因子1α(hypoxia-inducible factor-1 alpha,HIF-1α)及缺氧特异性微小RNA-210(microRNA-210,miR-210)的影响,探讨肿瘤细胞在低氧环境下miR-210表达的变化情况。方法:HepG2细胞经体外常规培养后,用体积分数为1%的氧气分别培养HepG2细胞6、12、18和24h。应用实时荧光定量RT-PCR(real-time quantitative reverse transcriptionPCR,qRT-PCR)和蛋白质印迹法分别检测细胞中HIF-1αmRNA和蛋白的表达变化,同时用茎环RT-PCR检测miR-210的表达变化,分析HIF-1α与miR-210的表达关系。染色质免疫共沉淀PCR(chromatin immunoprecitation PCR,ChIP-PCR)分析HIF-1α与miR-210启动子结合的可能性。结果:低氧培养6～12h,HIF-1αmRNA的表达量存在明显改变(P<0.05),而其蛋白的表达量从低氧培养开始直至24h均有明显变化(P<0.005);与此同时,miR-210的表达量在低氧培养6～18h时出现明显改变(P<0.01),且与HIF-1α蛋白表达存在明显的线性相关(R=0.795,P=0.037)。进一步的ChIP-PCR实验表明,HIF-1α在HepG2细胞内可与miR-210启动子区近转录区的低氧反应元件(hypoxia response element,HRE)结合,从而发挥转录调控作用。结论:适度的低氧环境可能影响miR-210表达,其机制可能是通过HIF-1α结合其启动子来调节实现的。Objective：To investigate the potential effects of hypoxia on the expressions of hypoxia-inducible factor-1 alpha（HIF-1α） and microRNA-210（miR-210） in hepatocellular carcinoma HepG2 cells,and to explore the possible mechanism of the change of miR-210 expression in tumor cells under the hypoxia microenvironment.Methods：HepG2 cells were routinely cultured in vitro,and then exposed to 1% oxygen for 6,12,18 and 24 h.Real-time quantitative reverse transcription PCR（qRT-PCR） and Western blotting were used to detect the expression levels of HIF-1α mRNA and protein.The expression of miR-210 in HepG2 cells under hypoxia condition at different time was assessed by stem-loop RT-PCR.The correlation between the expression of miR-210 and HIF-1α was analyzed.Finally,the chromatin immunoprecitation PCR（ChIP-PCR） was used to verify the hypothesis that miR-210 was the target of HIF-1α in HepG2 cells.Results：The change of HIF-1α mRNA expression in HepG2 cells was obvious under hypoxia condition for 6-12 h（P〈0.05）,whereas the HIF-1α protein expression was significantly changed from the beginning of hypoxic culture through 24 h（P〈0.005）.Meanwhile,the expression of miR-210 showed great change under hypoxia condition for 6-18 h（P〈0.01）,which was in a linear correlation with the expression of HIF-1α（R=0.795,P=0.037）.Moreover,ChIP-PCR assay showed that HIF-1α protein was bound to hypoxia response element（HRE） near transcriptional site in promoter region of miR-210 gene in HepG2 cells.Conclusion：The moderate hypoxia may affect the expression level of miR-210,and the possible mechanism may involve the HIF-1α protein bound to the promoter region of miR-210 gene.

关 键 词：癌 肝细胞 微小RNA类 低氧 缺氧诱导因子1 α亚单位 染色质免疫沉淀 

分 类 号：R735.7[医药卫生—肿瘤]