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

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（non-small 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，N-cadherin、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。

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

Objective:To 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 conducted 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-regulated，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，β-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 revealed 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，β-catenin，Vimentin and Slug.Conclusion:These results suggest that miR-29a could promote TGF-β1-induced EMT in NSCLC cells through down-regulating PTEN expression.