上调miR-125a对肺癌A549细胞凋亡的影响及其机制研究

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。

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

Objective 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 eukary- otic expression plasmid pGenesil-1 to construct recombinant plasmid pGenesil-miR-125a. Meanwhile, a control plasmid pGenesil-con- trol 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 An- nexin 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 apoptotie rate of A549-miR-125 a group was （31.04±2.48 ）% , higher than （6. 91±0. 72 ）% of A549-eontrol 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-eontrol 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.