MAP3K3基因过表达通过多条信号通路激活CREB促进人肺腺癌细胞增殖及上皮-间质转化

Overexpression of MAP3K3 activates CREB through multiple signaling pathways to promote the proliferation and epithelial-mesenchymal transition of human lung adenocarcinoma cells

目的通过体外干预有丝分裂原活化蛋白激酶激酶激酶3(MAP3K3)基因表达研究其对人肺腺癌细胞增殖、侵袭以及上皮-间质转化(EMT)的影响,探讨MAP3K3在肺腺癌发生、发展中的作用及可能的机制。方法使用慢病毒转染构建稳定过表达MAP3K3的人肺腺癌细胞株H1299、A549,通过克隆形成实验、划痕实验及Transwell实验,检测细胞增殖、迁移及侵袭行为的变化;Western blot实验检测EMT相关蛋白波形蛋白(Vimentin)、钙黏蛋白E(E-cadherin)和钙黏蛋白N(N-cadherin)表达水平,检测MAP3K3下游信号分子AKT、mTOR、ERK1/2、JNK1/2及其磷酸化蛋白表达水平,检测过表达和敲低MAP3K3的H1299细胞p-CREB蛋白表达水平变化。结果过表达MAP3K3基因后,H1299和A549细胞的增殖、迁移及侵袭能力明显增强,细胞的上皮表型分子E-cadherin下调,间质表型分子Vimentin和N-cadherin上调,p-AKT、p-mTOR、p-ERK1/2、p-JNK1/2以及p-CREB蛋白表达上调;使用shRNA稳定敲低MAP3K3后p-CREB蛋白表达明显下调。结论人肺腺癌细胞株H1299和A549体外过表达MAP3K3基因后,可增强细胞增殖、迁移及侵袭能力,促进细胞EMT,其机制可能与MAP3K3通过激活多个下游信号通路,磷酸化激活CREB(环磷腺苷效应元件结合蛋白)有关,此研究可为基于该信号通路的新型靶向药物开发提供一些实验依据。

Objective To investigate the influence of MAP3K3 on invasion,metastasis and epithelial-mesenchymal transition(EMT)of lung adenocarcinoma based on in vitro experiment.Methods Lentivirus transfection was employed to construct stable lung adenocarcinoma strains of H1299 and A549 overexpressing MAP3K3.With the help of clone formation experiment,scratch experiment and transwell experiment,the effect of overexpression of MAP3K3 gene on cell proliferation,migration and invasion behavior was detected.After the overexpression of MAP3K3 gene in H1299 and A549 lung adenocarcinoma cells,Western Blot experiment was conducted to detect the changes of the markers of epithelial mesenchymal transformation(Vimentin,E-cadherin,and N-cadherin).The changes of the downstream signal molecules,such as AKT/p-AKT,mTOR/p-mTOR,ERK/p-ERK,JNK/p-JNK and p-CREB,were further detected by Western Blot in H1299 and A549 lung adenocarcinoma cells with overexpression or knockdown of MAP3K3 gene.Results After overexpression of MAP3K3 in lung adenocarcinoma cell lines A549 and H1299,the cell proliferation,migration and invasion ability were enhanced.Overexpression of MAP3K3 down-regulated the expression of E-cadherin and up-regulated the expression of Vimentin and N-cadherin in A549 and H1299 lung adenocarcinoma cells.The expression of p-AKT,p-mTOR,p-ERK1/2,p-JNK1/2,and p-CREB were elevated after overexpression of MAP3K3.MAP3K3 knock-down by shRNA resulted in decrease of p-CREB.Conclusion Overexpression of MAP3K3 in vitro can promote the proliferation,migration,invasion and cell epithelial-mesenchymal transition in human adenocarcinoma cells.The mechanism may be related to MAP3K3 activating multiple downstream signaling pathways and phosphorylation to activate CREB(cAMP-response element binding protein),which provide an experimental basis for new targeted drugs based on MAP3K3 signaling pathway of lung cancer.