低氧微环境下siRNA靶向沉默HIF-2α基因对前列腺癌PC-3细胞增殖和凋亡的影响  

Effects of Silencing HIF-2α Gene by siRNA on Human Prostate Cancer PC-3 Cell Apoptosis And Proliferation Under Hypoxic Conditions

作　　者：陈剑晖[1] 李晓帆[2] 穆细院[1] 李永生[1] 蔡伟忠[1] 

机构地区：[1]福建医科大学附属协和医院泌尿外科,福州350001 [2]福建医科大学附属协和医院血液科,福建省血液病研究所,福州350001

出　　处：《福建医科大学学报》2017年第5期275-281,共7页Journal of Fujian Medical University

基　　金：福建省卫生厅青年基金(2010-2-17);福建省青年拔尖创新人才及福建省杰出青年科研人才培育计划(JA14130)

摘　　要：目的观察体外低氧条件下缺氧诱导因子-2α(HIF-2α)在人前列腺癌细胞PC-3中的表达情况,并研究siRNA沉默HIF-2α基因对前列腺癌PC-3细胞生长及凋亡的影响。方法在体外培养的PC-3细胞中加入化学诱导剂CoCl_2建立低氧模型,采用逆转录PCR(RT-PCR)和Western印迹法检测CoCl_2诱导HIF-2αmRNA和蛋白表达的时-效和量-效关系。合成HIF-2αsiRNA片段并转染前列腺癌PC-3细胞,采用RT-PCR及Western印迹法检测HIF-2αsiRNA对HIF-2α基因表达的影响,采用MTT法检测转染HIF-2αsiRNA后对PC-3细胞生长的抑制情况,采用流式细胞仪检测RNA干扰对PC-3细胞凋亡的影响。结果 (1)低氧可诱导PC-3细胞的HIF-2αmRNA表达,与常氧组比较,低氧12,24及48h组的HIF-2αmRNA和蛋白表达量逐渐升高(P<0.05)。100μmol/L的CoCl_2作用48h可作为最佳前列腺癌细胞PC-3低氧模型。(2)低氧+HIF-2αsiRNA干扰组的HIF-2αmRNA和蛋白表达水平明显低于低氧+阴性对照组和低氧组(P<0.05),而低氧+阴性对照组与低氧组的HIF-2αmRNA和蛋白表达水平无显著性差异(P>0.05)。低氧+HIF-2αsiRNA干扰组细胞增殖受抑制、细胞凋亡增加(P<0.05),而常氧组、低氧组、低氧+阴性对照组细胞增殖活力和凋亡差别均无统计学意义(P>0.05)。结论低氧可促进前列腺癌PC-3细胞表达HIF-2α逐渐上调,siRNA干扰HIF-2α可以抑制低氧状态下前列腺癌PC-3细胞HIF-2α基因的表达,达到抑制细胞增殖、促进细胞凋亡的目的。阻断HIF-2α信号通路可作为激素非依赖前列腺癌治疗的新策略。Objective To observe the expression of hypoxia-inducible factor-2 alpha（HIF-2α ）in human prostate cancer PC-3 cell under hypoxia,and investigate the effects of silencing HIF-2α gene by siRNA on PC-3 cell apoptosis and proliferation under hypoxia. Methods Tumor hypoxia was induced by CoCl2 chemical hypoxia method. RT-PCR and Western-blot was performed to detect the expression of HIF-2α mRNA and protein in human prostate cancer PC-3 cells,and the relations of the quantity-efficiency and the time-efficiency were analyzed. siRNA was chemically synthesized and transfected into PC-3 cells. The expressions of HIF-2α in hypoxia micro-environment were respectively detected by RT-PCR and Western-blot. The effects of apoptosis and proliferation were detected by FCM and MTT assay. Results（1）Hypoxia improved HIF-2α expression in PC-3 cells in a time-dependent manner. Compared with the normal oxygen group,the HIF-2α mRNA and protein expression level was significantly higher in 12 h-,24 h-,and 48 h-group（P〈0.05）. The best model of hypoxia was 48 h-group induced by 100μmol/L CoCl2.（2）The levels of mRNA and protein of HIF-2α were suppressed significantly after being treated with HIF-2α siRNA in PC-3 cells（P〉0.05）,but there was no significant difference in cell proliferation and apoptosis between CoCl2 negative control group and CoCl2 group（P〉0.05）. The proliferation potential and apoptosis in PC-3 cells were changed significantly after transfecting HIF-2α siRNA（P〉0.05）,but there was no significant difference among normal oxygen group,CoCl2 groups,and CoCl2 negative control group（P〉0.05）. Conclusion HIF-2α is over-expressed in human prostate cancer cell PC-3 after CoCl2 treatment. HIF-2α siRNA effectively reduces the HIF-2α mRNA expressions in PC-3 cells. Tansfecting HIF-2α siRNA inhibits PC-3 cells proliferation and increases PC-3 cells apoptosis. Blocking HIF-2α signal pathway can be a potential strategy in the treatment of androgen independent prostate cancer.

关 键 词：前列腺肿瘤 RNA干扰 基因 细胞增殖 细胞凋亡 

分 类 号：R737.25[医药卫生—肿瘤]