靶向egfr基因rna干扰对人卵巢癌skov3细胞-第三军医大学学报.docVIP

靶向EGFR基因RNA干扰对人卵巢癌SKOV3细胞增殖的抑制作用 郭启帅，黄 曦2，吴永忠3，李少林1 (重庆医科大学: 1放射医学教研室,2附属第一医院血液科,重庆400016；3重庆市肿瘤研究所 重庆 400030) 摘要： 目的 利用RNA干扰技术下调EGFR基因的表达，观察其对卵巢癌SKOV3细胞增殖的影响。方法 构建针对EGFR基因的siRNA真核表达载体，转染入卵巢癌SKOV3细胞中，并筛选稳定表达siRNA的转化克隆，RT-PCR及Western blot分别检测EGFR mRNA和蛋白表达，流式细胞仪检测细胞周期和凋亡,克隆形成实验及MTT法检测细胞克隆形成率及细胞体外的增殖情况。结果 成功构建表达质粒pGenSil-HK、pGenSil-EGFR1和pGenSil-EGFR2，并转染SKOV3细胞，通过G418筛选后获得稳定转染克隆，RT-PCR及Western blot分析表明转染pGenSil-EGFR1、pGenSil-EGFR2后的细胞EGFR mRNA和蛋白表达均受到了明显抑制，EGFR mRNA抑制率分别为41.87％和68.07％，蛋白表达抑制率分别为45.21％和70.25％。与未转染组和pGenSil-HK组相比，pGenSil-EGFR2组细胞凋亡比例显著增加，G1 期细胞增多，而S期细胞减少（P＜0.05）；转染pGenSil-HK组克隆形成率为(0.82±0.03)，转染pGenSil-EGFR2组克隆形成率为（0.61±0.04），两者比较差异有统计学意义（P＜0.05），MTT显示细胞培养36 h后，pGenSil-EGFR2组的细胞生长均显著低于未转染组和转染pGenSil-HK组（P＜0.05）。结论 靶向EGFR基因RNA干扰能够阻抑卵巢癌SKOV3细胞中的EGFR表达，诱导细胞凋亡、调控细胞周期再分布、抑制细胞增殖。 关键词: RNA干扰；表皮生长因子受体； SKOV3细胞；细胞周期；凋亡 中图法分类号： R737.31 文献标识码： A Effect of RNA interference targeting EGFR gene on proliferation of human ovarian carcinoma cell line SKOV3 GUO Qi-Shuai1，HUANG Xi 2，WU Yong-Zhong3，LI Shao-Lin1 (1Department of Radiation Medicine，2Department of Hematology，The First Affiliated Hospital，Chongqing Medical University，Chongqing 400016，China；3Cancer institute of Chongqing，Chongqing 400030，China) A： 0bjectiveTo explore the influence of down-regulating EGFR expression by RNA interference on the proliferation of ovarian carcinoma SKOV3 cells. Methods The recombinant eukaryotic expression plasmids pGenSil-HK、pGenSil-EGFR1 and pGenSil-EGFR2 were constructed and transfected into SKOV3 cells. The mRNA and protein expressions of EGFR in SKOV3 cells were detected by RT-PCR and Western-Blotting,respectively. The cell cycle and apoptosis were evaluated by flow cytometry. The proliferation of SKOV3 cells in vitro was determined by clone formation assay and MTT assay. Results we successfully constructed the recombinant eukaryotic expression plasmids pGenSil-HK、pGenSil-EGFR1 and pGenSil-EGFR2 and transfected into SKOV3 cells. Three cell clones were screened