过表达miR-126对人肺癌细胞体外生长的抑制作用和初步机制研究

摘要

目的:探讨过表达miR-126对人肺癌95D细胞体外生长的影响.方法:利用PCR技术成功构建pcDNA3.1(-)-pri-miR-126载体(命名为p-miR-126),将p-miR-126重组质粒体外瞬时转染人肺癌95D细胞,TaqMan探针法检测miR-126的相对表达水平,CCK-8(cell counting kit-8)法和划痕法分别检测95D细胞的增殖、迁移能力.Western Blot检测95D细胞中蛋白AKT和磷酸化AKT(phosphorylation AKT,p-AKT)的表达情况.结果:成功构建miR-126真核表达载体,且该载体可上调miR-126表达(P＜0.05),抑制95D细胞的生长和迁移能力(P＜0.05).Western Blot结果表明细胞中磷酸化AKT水平显著降低(P＜0.05).结论:通过上调miR-126的表达水平可有效抑制95D细胞的体外生长,其作用机制有可能与AKT信号通路的变化有关,但其具体机制仍需后续深入研究.%Objective:To explore the effect of miR-126 overexpression on the growth of human lung cancer 95D cells in vitro.Methods:pcDNA3.1 (-)-pri-miR-126 vector (named p-miR-126) was successfully constructed by using PCR technique.p-miR-126 was transiently transfected into 95D cells and the expression level of miR -126 was determined by TaqMan probe before and after transfection.The proliferation ability of 95D cells were detected by CCK-8 assay and the migration of 95D cells in vitro were determined by scratch assay.In addition,the level of AKT and phosphorylated AKT were analyzed by Western blot assay.Results:pcDNA3.1-miR-126 eukaryotic expression vector was successfully constructed which can upregulation of miR-126 expression (P ＜ 0.05).The proliferation and migration of 95 D calls in vitro was inhibited (P ＜ 0.05).Meanwhile,the level of phosphorylation AKT was significantly decreaced.Conclusion:Overexpression of miR-126 could inhibit the growth of human lung cancer cells effectively in vitro,which may be related to the changes of AKT signaling pathway.