临床肿瘤学杂志

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上调miR-125a对肺癌A549细胞凋亡的影响及其机制研究

胡金华,吴玉泉,陈清勇,赵园园,焦德敏   

  1. 310013 杭州 解放军第一一七医院内科
  • 收稿日期:2012-11-08 修回日期:2012-12-25 出版日期:2013-03-31 发布日期:2013-03-31
  • 通讯作者: 吴玉泉

Effects of up-regulated expression of miR-125a on cell apoptosis of human lung cancer A549 cells and possible mechanisms

HU Jinhua,WU Yuquan,CHEN Qingyong,ZHAO Yuanyuan,JIAO Demin.   

  1. epartment of Internal Medicine,the 117th Hospital of PLA,Hangzhou 310013,China
  • Received:2012-11-08 Revised:2012-12-25 Online:2013-03-31 Published:2013-03-31

摘要: 目的 探讨上调microRNA-125a(miR-125a)对肺癌A549细胞凋亡的影响及可能机制。方法 人工合成miR-125a基因序列,将miR-125a基因克隆至真核表达载体pGenesil-1质粒,构建重组质粒pGenesil-miR-125a,同时设计并构建阴性对照质粒pGenesil-control。将以上载体经脂质体介导转染肺癌A549细胞并分为3组:成功转染pGenesilmiR-125a的A549细胞(A549-miR-125a组),成功转染pGenesil-control的A549细胞(A549-control组),以及未进行转染的A549细胞(A549组)。采用实时定量PCR技术(QPCR)检测各组细胞的miR-125a表达水平,Annexin V-FITC/PI双染流式细胞术检测细胞凋亡率,免疫印迹法检测p53基因的蛋白水平。结果 经酶切和测序鉴定证明pGenesil-miR-125a重组质粒构建成功。A549-miR-125a组的miR-125a水平为2.72±0.41,高于A549-control组的0.97±0.16和A549组的0.96±0.11(P<0.01);A549-miR-125a组的细胞凋亡率为(31.04±2.48)%,高于A549-control组的(6.91±0.72)%和A549组的(6.73±0.56)%(P<0.05);A549-miR-125a组的p53蛋白水平为3.91±0.46,高于A549-control组的1.01±0.06和A549组的0.99±0.04(P<0.01)。结论 上调miR-125a可促进肺癌细胞的凋亡,提示其在肺癌的发生、发展中有重要作用,可能的机制是上调p53的蛋白表达。

Abstract: bjective To explore the effect of up-regulated expression of microRNA-125a(miR-125a) on cell apoptosis of human lung cancer A549 cells and possible mechanisms. Methods The miR-125a sequence was synthesized and cloned into eukaryotic expression plasmid pGenesil-1 to construct recombinant plasmid pGenesil-miR-125a.Meanwhile,a control plasmid pGenesil control was constructed. The above plasmids were transfected into human lung cancer A549 cells using liposomes and divided into three groups: A549 cells transfected with pGenesilmiR-125(A549-miR-125a group),A549 cells transfected with pGenesil control(A549-control group)and A549 cells without transfection(A549). Quantitative realtime polymerase chain reaction(QPCR) was used to measure the levels of miR-125a(normalized to U6 mRNA levels) after transfection. The percentage of apoptosis was determined by Annexin V-FITC/PI double staining with flow cytometry analysis. Western blot was employed to analyze the protein levels of p53. Results The recombinant plasmid pGenesil-miR-125a was verified by restriction analysis and DNA sequencing. The level of miR-125a in A549-miR-125a group was 2.72±0.41,higher than 0.97±0.16 of A549-control group and 0.96±0.11 of A549 group(P<0.01). The apoptotic rate of A549-miR-125a group was(31.04±2.48)%,higher than (6.91±0.72)% of A549-control group and (6.73±0.56)% of A549 group(P<0.05). The level of p53 protein in A549-miR-125a group was 3.91±0.46,higher than 1.01±0.06 of A549 control group and 0.99±0.04 of A549 group(P<0.01). Conclusion Up-regulated expression of miR-125a can promote the apoptosis of lung cancer A549 cells with the possible mechanism of the up regulation of p53.

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