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紫杉醇诱导HepG2肝癌细胞凋亡中miR-224沉默凋亡抑制因子5基因的作用
单长民* 孙业盈 李娟 王东 姚庆收 谭秀华 刘向勇
解剖学报 ›› 2015, Vol. 46 ›› Issue (4) : 521-527.
紫杉醇诱导HepG2肝癌细胞凋亡中miR-224沉默凋亡抑制因子5基因的作用
Apoptosis inhibitor 5 gene silenced by miRNA-224 in paclitaxel inducing HepG2 cells apoptosis
目的 探讨紫杉醇诱导肝癌HepG2 细胞凋亡时miRNA对基因的沉默的作用。 方法 用不同浓度紫杉醇处理肝癌HepG2细胞后，流式细胞术检测其细胞凋亡；通过实时定量PCR(Real-time PCR)测定miR-224和凋亡抑制因子5（api-5） mRNA的表达；用免疫组织化学二步法和免疫印迹法(Western blotting)检测API-5蛋白的表达。
结果 紫杉醇诱导HepG2细胞凋亡时，通过2-ΔΔCT法分析发现，除了0.5mg/L、1mg/L紫杉醇培养HepG2肝癌细胞24h miR-224不升反降外，其他浓度和处理时间miR-224表达均显示上调；0.5mg/L、1mg/L紫杉醇培养HepG2肝癌细胞24h api-5 mRNA表达稍微降低，0.5mg/L紫杉醇处理48h无增长，其他浓度和处理时间api-5 mRNA表达均上调；应用IPP v 5.1图像分析软件处理，经 t 检验分析发现，紫杉醇处理后API-5蛋白表达均下调（P<0.05）。 结论 上调的miR-224可能作用其靶基因api-5的表达，通过与api-5 mRNA的3’UTR结合阻止翻译的完成，从而对肝癌细胞的凋亡途径产生影响。
Objective To explore the gene silencing of microRNAs in paclitaxel inducing HepG2 cells apoptosis.Methods After treated with different concentrations of paclitaxel, apoptosis of human hepotoma cell line HepG2 was detected by flow cytometry, expressions of miR-224 and apoptosis inhibitors 5(api-5) mRNA were determined by Real-time PCR, the expression of API-5 protein was detected by immunohistochemical two steps method and Western blotting method. Results In paclitaxel inducing HepG2 cells apoptosis, the expressions of miR-224 in HepG2 treated with 0.5 mg/L, 1mg/L paclitaxel for 24hours decreased slightly, while those of the others increased obviously by analysis of 2-ΔΔCT method. The expressions of api-5 mRNA in HepG2 treated with 0.5 mg/L, 1mg/L paclitaxel for 24hours decreased slightly, while those of the others were up-regulated except that those of treatment with 0.5mg/L paclitaxel and the control was similar. By the treatment of Image Pro Plus v 5.1,it was found that the levels of API-5 protein were down-regulated with treatment of paclitaxel through the analysis of t-test（P<0.05）. Conclusion Increased miR-224 may affect the expression of its target gene api-5. Through the combination with api-5 mRNA 3’UTR,the completion of translation is prevented, which may impact on liver cancer cell apoptosis pathway.
紫杉醇 / 肝癌细胞HepG2 / 微小RNA-224 / 凋亡抑制因子5 / 实时定量聚合酶链反应 / 免疫印迹法
Paclitaxel / Human hepotoma cell line HepG2 / MicroRNA-224 / Apoptosis inhibitor 5 / Real-time PCR / Western blotting
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