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

doi:10.16098/j.issn.0529-1356.2015.04.013

解剖学报 ›› 2015, Vol. 46 ›› Issue (4): 521-527.doi: 10.16098/j.issn.0529-1356.2015.04.013

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^1* 孙业盈¹ 李娟²王东³ 姚庆收⁴ 谭秀华¹刘向勇⁵

1.滨州医学院医学遗传学教研室; 2.滨州医学院附属医院药剂科; 3.滨州医学院组织学与胚胎学教研室; 4.滨州医学院基因工程教研室; 5.滨州医学院细胞生物学教研室，山东滨州 256603

收稿日期:2014-11-17 修回日期:2015-03-10 出版日期:2015-08-06 发布日期:2015-08-06
通讯作者: 单长民 E-mail:674650785@qq.com
基金资助:
烟台市科技计划课题

Apoptosis inhibitor 5 gene silenced by miRNA-224 in paclitaxel inducing HepG2 cells apoptosis

SHAN Chang-min ^1*SUN Ye-ying¹ LI Juan² WANG Dong³ YAO Qing-shou⁴TAN Xiu-hua¹ LIU Xiang-yong⁵

1. Department of Medical Genetics，Binzhou Medical Universtiy; 2. Department of Pharmacy，Affiliated Hospital of Binzhou Medical Universtiy; 3. Department of Histology and Embryology，Binzhou Medical Universtiy; 4. Department of Gene Engineering，Binzhou Medical Universtiy; 5. Department of Cell Biology, Binzhou Medical Universtiy, Shandong Binzhou 256603, China

Received:2014-11-17 Revised:2015-03-10 Online:2015-08-06 Published:2015-08-06
Contact: SHAN Chang-min E-mail:674650785@qq.com

摘要/Abstract

摘要：

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。

关键词: 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

Abstract:

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.

Key words: Paclitaxel, Human hepotoma cell line HepG2, MicroRNA-224, Apoptosis inhibitor 5, Real-time PCR, Western blotting

单长民* 孙业盈李娟王东姚庆收谭秀华刘向勇. 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[J]. 解剖学报, 2015, 46(4): 521-527.

SHAN Chang-min* SUN Ye-ying LI Juan WANG Dong YAO Qing-shou TAN Xiu-hua LIU Xiang-yong. Apoptosis inhibitor 5 gene silenced by miRNA-224 in paclitaxel inducing HepG2 cells apoptosis[J]. AAS, 2015, 46(4): 521-527.

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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

Apoptosis inhibitor 5 gene silenced by miRNA-224 in paclitaxel inducing HepG2 cells apoptosis

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