miR-93通过靶向EphA4激活ERK通路促进非小细胞肺癌细胞的增殖和迁移

miR-93 activates ERK pathway to promote proliferation and migration of NSCLC cells via targeting EphA4

目的:探讨miR-93/Eph受体A4(EphA4)分子轴通过细胞外调节蛋白激酶(extracellular regulated protein kinases),ERK通路对非小细胞肺癌(non-small cell lung cancer,NSCLC)H460和H1299细胞增殖和迁移的影响。方法:用qPCR检测H460和H1299细胞中miR-93表达水平。分别在H460细胞中转染miR-93模拟物(mimics)和EphA4过表达质粒、在H1299细胞中转染miR-93抑制剂(inhibitor)后,用MTT、Transwell实验检测miR-93对转染细胞增殖和迁移的影响。用双荧光素酶报告基因实验验证miR-93与EphA4之间的靶向调控关系。用Western blotting检测细胞中增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)、EphA4、ERK和p-ERK蛋白的表达水平,用MTT、Transwell实验检测同时过表达miR-93和EphA4对H460细胞增殖和迁移的影响。结果:miR-93在H1299细胞中表达水平高于H460细胞(P<0.01)。过表达miR-93促进H460细胞增殖和迁移(均P<0.01),敲低miR-93抑制H1299细胞增殖和迁移(均P<0.01)。双荧光素酶报告基因实验证实miR-93靶向调控EphA4,过表达miR-93明显下调H460细胞中EphA4 mRNA和蛋白表达水平(均P<0.05),过表达miR-93通过靶向EphA4并激活ERK通路促进H460细胞的增殖和迁移(均P<0.01)。结论:miR-93促进NSCLC细胞增殖和迁移,其机制可能与靶向调控EphA4并激活ERK通路有关。

Objective: To investigate the effect of miR-93/EphA4(Eph receptor A4) axis on the proliferation and migration of nonsmall cell lung cancer(NSCLC) H460 and H1299 cells via regulating extracellular regulated protein kinases(ERK) pathway. Methods:The expression levels of miR-93 in H460 and H1299 cells was detected by qPCR. miR-93 mimics and EphA4 overexpression plasmids were transfected into H460 cells and miR-93 inhibitor was transfected into H1299 cells respectively, after which MTT assay and Transwell assay were used to detect the effects of miR-93 on proliferation and migration of transfected cells. The targeted regulatory relationship between miR-93 and EphA4 was verified by Dual-luciferase reporter gene assay. The expression levels of PCNA(proliferating cell nuclear antigen), EphA4, ERK and p-ERK were detected by Western blotting. The effects of simultaneous overexpression of miR-93 and EphA4 on proliferation and migration of H460 cells were detected by MTT assay and Transwell assay. Results: The expression of miR-93 in H1299 cells was higher than that in H460 cells(P<0.01). Overexpression of miR-93 promoted proliferation and migration of H460 cells(all P<0.01), and knockdown of miR-93 inhibited proliferation and migration of H1299 cells(all P<0.01). The Dualluciferase reporter gene assay confirmed that miR-93 could target EphA4. Overexpression of miR-93 down-regulated the mRNA and protein expression levels of EphA4(all P<0.05), and promoted proliferation and migration of H460 cells through targeted regulation of EphA4 and activation of ERK pathway(all P<0.01). Conclusion: miR-93 promotes the proliferation and migration of NSCLC cells,and its mechanism may be related to the targeted regulation of EphA4 and activation of the ERK pathway.