MAPK1基因沉默对肺癌A549细胞增殖、凋亡、迁移和侵袭能力的影响

Effect of MAPK1 Gene Silencing on Proliferation,Apoptosis,Migration and Invasion of Lung Cancer A549 Cells

目的探讨丝裂原活化蛋白激酶1(mitogen-activated protein kinases1,MAPK1)基因沉默对肺癌A549细胞的增殖、凋亡、迁移和侵袭能力的影响。方法利用CRISPR/CAS9基因编辑技术构建MAPK1沉默细胞系,采用Real-time PCR和Western blot检测沉默MAPK1后mRNA和蛋白表达水平。将MAPK1沉默载体与空载体转染至肺癌A549细胞中,并分为MAPK1沉默组、空载体组和空白组。对各组的肺癌A549细胞进行培养,利用CCK-8实验检测细胞增殖能力;平板集落形成实验检测细胞集落形成能力;平板划痕实验和Transwell实验检测细胞迁移和侵袭能力;流式细胞术检测细胞凋亡和周期情况以及Western blot实验检测MAPK1沉默后ERK/mTOR通路蛋白表达。结果Real-time PCR和Western blot结果表明成功构建肺癌A549细胞MAPK1沉默细胞系。MAPK1沉默使肺癌A549细胞增殖能力减弱(P<0.05),迁移和侵袭能力降低(P<0.05),细胞凋亡率增加且细胞周期被阻滞于S期(P<0.05)。MAPK1沉默后,ERK/mTOR通路信号分子mTOR、PRAS40、EIF4EBP1、MEK2的总蛋白和磷酸化蛋白表达均降低(P均<0.05)。结论沉默MAPK1可抑制肺癌A549细胞增殖、迁移、侵袭,促进细胞凋亡,阻滞细胞周期。

Objective To investigate the effect of mitogen-activated protein kinases1(MAPK1)gene silencing on the proliferation,apoptosis,migration and invasion of human lung cancer A549 cells.Methods The MAPK1 gene silencing cell line in human lung cancer cell A549 was constructed by the CRISPR/CAS9 system,Real-time PCR and Western blot experiments were used to detect the expression levels of MAPK1 mRNA and protein after gene silencing.The MAPK1 silencing vector and empty vector were transfected into lung cancer A549 cells and divided into the MAPK1 silencing group,the empty vector group and the blank control group.The lung cancer A549 cells were cultured in blank groups,the empty vector group,and the MAPK1 silencing group.The proliferation of cells was detected by CCK-8.The colony forming ability was detected by cell clone experiment.The cell scratch assay and the Transwell assay were used to detect cell migration and invasion.The cell apoptosis and the cell cycle were detected by Flow cytometry,and the changes of ERK/mTOR pathway protein relative expression after MAPK1 silencing were detected by Western blot.Results Real-time PCR and Western blot showed that the MAPK1 silencing cell line of lung cancer A549 cells was successfully constructed.MAPK1 silencing decreased the cell proliferation(P<0.05),migration and invasion ability(P<0.05),increased the apoptosis rate and arrested the cell cycle in S phase(P<0.05).After MAPK1 silencing,the total and phosphorylated protein expression of mTOR,PRAS40,EIF4EBP1 and MEK2 were decreased in ERK/mTOR pathway(P all<0.05).Conclusion MAPK1 silencing can inhibit the proliferation,migration and invasion,promote cell apoptosis and arrest cell cycle of human lung cancer A549 cells.