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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
引用本文:梁 媛1,冯洋洋2,李琳琳1,沈晓宇1,辛 田1,赵雨薇1,马 锐1. 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[J]. 现代肿瘤医学, 2018, 0(5): 653-659. DOI: 10.3969/j.issn.1672-4992.2018.05.002
作者姓名:梁 媛1  冯洋洋2  李琳琳1  沈晓宇1  辛 田1  赵雨薇1  马 锐1
作者单位:1.中国医科大学肿瘤医院 辽宁省肿瘤医院内四科,辽宁 沈阳 1100422.中国医科大学基础医学院病理学与病理生理学科,辽宁 沈阳 110122
基金项目:辽宁省自然科学基金项目(编号:20170540562)
摘    要: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。

关 键 词:非小细胞肺癌  miR-29a  PTEN  上皮间质转化

MicroRNA-29a promotes epithelial-mesenchymal transition in non-small cell lung cancer through down-regulating PTEN gene expression
Liang Yuan1,Feng Yangyang2,Li Linlin1,Shen Xiaoyu1,Xin Tian1,Zhao Yuwei1,Ma Rui1. MicroRNA-29a promotes epithelial-mesenchymal transition in non-small cell lung cancer through down-regulating PTEN gene expression[J]. Journal of Modern Oncology, 2018, 0(5): 653-659. DOI: 10.3969/j.issn.1672-4992.2018.05.002
Authors:Liang Yuan1  Feng Yangyang2  Li Linlin1  Shen Xiaoyu1  Xin Tian1  Zhao Yuwei1  Ma Rui1
Affiliation:1.Department of No.4 Internal Medicine,Cancer Hospital of China Medical University,Liaoning Cancer Hospital,Liaoning Shenyang 110042,China;2.Institute of Pathology and Pathophysiology,College of Basic Medical Science,China Medical University,Liaoning Shenyang 110122,China.
Abstract: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.
Keywords:non-small cell lung cancer   microRNA-29a   PTEN   epithelial-mesenchymal transition
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