MicroRNA-29a靶向抑制PTEN基因诱导非小细胞肺癌细胞上皮间质转化的机制研究

MicroRNA-29a promotes epithelial-mesenchymal transition in non-small cell lung cancer through down-regulating PTEN gene expression

目的:探讨microRNA-29a(miR-29a)及其靶蛋白PTEN在TGF-β1诱导非小细胞肺癌(nonsmall cell lung cancer,NSCLC)细胞上皮间质转化(epithelial-mesenchymal transition,EMT)中的作用机制。方法:选择A549细胞经终浓度为10 ng/ml TGF-β1诱导48 h后,分为Blank组(不转染任何序列)、阴性对照(negative control,NC)组(转染阴性对照序列)、IN组(转染miR-29a inhibitors)、siRNA组(转染PTEN-siRNA)和IN+siRNA组(共转染miR-29a inhibitors和PTEN-siRNA)。普通显微镜观察各组细胞形态学变化;免疫荧光检测各组细胞中E-cadherin表达水平;qRT-PCR法检测EMT相关因子及PTEN mRNA表达水平;Western blotting法检测转染后EMT相关因子、PTEN、Akt和p-Akt蛋白的表达;划痕实验检测各组细胞迁移能力。构建裸鼠移植瘤模型,观察肿瘤生长,免疫组化检测裸鼠肿瘤组织中PTEN及EMT相关因子蛋白表达水平。结果:A549细胞转染miR-29a inhibitors后TGF-β1诱导细胞的上皮间质转化显著受到抑制,Ncadherin、Vimentin及Slug的mRNA和蛋白表达水平在IN组中显著低于Blank组和NC组,但在siRNA组和IN+siRNA组中显著上调(均P<0.05)。与Blank组和NC组相比,IN组PTEN mRNA和蛋白表达水平明显升高,且p-Akt的表达显著降低,细胞迁移率显著下降,而siRNA组和IN+siRNA组PTEN mRNA和蛋白表达明显降低,p-Akt的表达显著上升,细胞迁移率显著升高(均P<0.05)。裸鼠移植瘤实验结果显示与Blank组和NC组相比,IN组肿瘤生长较慢,重量降低,E-cadherin和PTEN蛋白表达显著升高,N-cadherin、β-catenin、Vimentin、Slug蛋白表达显著降低(均P<0.05)。结论:TGF-β1能诱导NSCLC细胞发生EMT,且能上调miR-29a并抑制PTEN的表达水平;抑制miR-29a的表达水平可能通过上调靶基因PTEN,促进Akt磷酸化,抑制EMT的发生。

ObjectiveTo explore the effects of microRNA -29a （miR -29a） and PTEN/Akt signaling pathway on TGF -β1 - induced epithelial - mesenchymal transition （ EMT） in non - small cell lung cancer （ NSCLC）. Methods：After 48 h of treatment with TGF -β1 （10 ng/ml）,the A549 cells were divided into the blank,negative control （NC） ,miR - 29a inhibitors （IN） , PTEN - siRNA and miR - 29a inhibitors ＋ PTEN - siRNA groups. EMT related factors and PTEN expressions were detected by qRT - PCR assay and Western blotting. The scratch test was conduc-ted to observe cell migration. Xenograft tumor model in nude mice was used to evaluate the effects of miR - 29a on EMT of NSCLC cells in vivo. Results ： In NSCLC tissues, the expressions of miR - 29a, Akt and p - Akt were up - reg-ulated, while PTEN expression was down - regulated. Gene and protein expressions of E - cadherin and PTEN in the miR - 29a inhibitors group were higher than the blank, NC, PTEN - siRNA and miR - 29a inhibitors ＋ PTEN - siRNA groups,while the expressions of N - cadherin, p - catenin, Vimentin and Slug were higher than other groups. miR - 29a inhibitors significantly inhibit cell migration and invasion in NSCLC cell lines. In vivo xenograft experiment re-vealed that miR - 29a inhibitor inhibits the growth of transplanted tumor through up - regulating E - cadherin and PTEN expressions and down - regulating the expressions of N - cadherin, p - catenin, Vimentin and Slug. Conclu-sion ：These results suggest that miR -29a could promote TGF -β1 - induced EMT in NSCLC cells through down - regulating PTEN expression.