miR-105-5p靶向MAPK1抑制非小细胞肺癌细胞A549的生长、运动和上皮间质转化

miR-105-5p inhibits growth,movement and epithelial-mesenchymal transition of non-small cell lung cancer cell line A549 by targeting MAPK1

目的:探究miR-105-5p靶向MAPK1对非小细胞肺癌(NSCLC)细胞A549的生长、运动和上皮间质转化(EMT)的影响。方法:利用Lipofectamine 2000将mimic-NC、miR-105-5p mimic、MAPK1质粒分别或联合转染至各组A549细胞;qRT-PCR检测miR-105-5p和MAPK1 mRNA表达水平,利用软件预测miR-105-5p靶向关系,并利用荧光素酶实验验证;MTT法检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Transwell检测细胞侵袭能力,划痕实验检测细胞迁移能力,Western blot检测MPAK1、Ki67、PCNA、Bcl-2、Bax、Caspase-3、Caspase-9、E-cadherin及N-cadherin表达水平;皮下注射A549细胞构建肺癌移植瘤模型,30 d后移植瘤称重,并用Western blot检测MPAK1、KI67、Bax、Caspase-3、E-cadherin蛋白表达情况。结果:miR-105-5p在NSCLC A549细胞低表达,miR-105-5p与MAPK1在3′UTR区存在结合位点,过表达miR-105-5p抑制MAPK1表达;miR-105-5p过表达后,NSCLC A549细胞的增殖速度及MPAK1、Ki67、PCNA和Bcl-2表达量降低,细胞凋亡率及Bax、Caspase-3和Caspase-9表达量升高,侵袭细胞数目和伤口愈合率减少,E-cadherin表达上调、N-cadherin表达明显下调,MAPK1过表达可逆转miR-105-5p过表达引起的改变。miR-105-5p过表达明显减轻移植瘤重量,下调移植瘤细胞中MPAK1、Ki67、Bax和Caspase-3表达,上调E-cadherin表达。结论:miR-105-5p靶向MAPK1抑制NSCLC细胞A549的生长、运动和EMT。

Objective:To investigate effect of miR-105-5p on growth,movement and epithelial-mesenchymal transition of non-small cell lung cancer(NSCLC)cell line A549 by targeting MAPK1.Methods:Mimic-NC,miR-105-5p mimic and MAPK1 plasmids were transfected into each group of A549 cells by Lipofectamine 2000.Expression levels of miR-105-5p and MAPK1 mRNA were detected by qRT-PCR.miR-105-5p targeting relationship was predicted by software and verified by luciferase assay.Cell proliferation was detected by MTT assay,apoptosis was detected by flow cytometry,cytometry invasive ability of cells was detected by Transwell,cell migration ability was detected by scratch test.Expression levels of MAPK1,Ki67,PCNA,Bcl-2,Bax,Caspase-3,Caspase-9,E-cadherin and N-cadherin were detected by Western blot.The lung cancer xenograft model was constructed by subcutaneous injection of A549 cells,tumor weight was transplanted 30 d later,and expressions of MAPK1,Ki67,Bax,caspase-3 and E-cadherin were detected by Western blot.Results:miR-105-5p was down-regulated in NSCLC A549 cell,and there was a binding site with MAPK1 in the 3′UTR region,and overexpression of miR-105-5p inhibited MAPK1 expression.After overexpression of miR-105-5p,cell proliferation rate and expression levels of MAPK1,Ki67,PCNA and Bcl-2 in NSCLC A549 cells were decreased,apoptotic rate and expression levels of Bax,Caspase-3 and Caspase-9 were increased,number of invasive cells and rate of wound healing were reduced,the expression of E-cadherin was significantly up-regulated and expression of N-cadherin was down-regulated,MAPK1 overexpression reversed the changes caused by miR-105-5p overexpression.Overexpression of miR-105-5p significantly reduced the weight of transplanted tumors,down-regulated the expressions of MAPK1,Ki67,Bax and caspase-3 in transplanted tumor cells,and up-regulated expression of E-cadherin.Conclusion:miR-105-5p inhibits growth,movement and EMT of NSCLC cell line A549 by targeting MAPK1.