MicroRNA-433在非小细胞肺癌A549细胞增殖及迁移中的作用

Role of MiRNA-433 in proliferation and migration of A549 cells

目的研究microRNA-433(miR-433)对人非小细胞肺癌(NSCLC)A549细胞增殖及迁移的作用。方法采用实时聚合酶链反应(real-time PCR)检测miR-433在A549、NCI-H1299、NCI-H358以及人肺成纤维细胞系HFL1中的表达。不同浓度mi R-433 mimic转染A549细胞,选取最佳浓度进行后续实验;MTT法检测细胞增殖情况;Transwell检测细胞迁移情况;双荧光素酶报告基因法检测mi R-433与p21活化激酶4(PAK4)的靶向关系;Western blotting检测PAK4、单丝氨酸蛋白激酶1(LIMK1)、磷酸化单丝氨酸蛋白激酶1(p-LIMK1)、丝切蛋白(Cofilin)和磷酸化丝切蛋白(p-Cofilin)的表达。结果与HFL1比较,mi R-433在3种NSCLC中的表达均降低。过表达miR-433 48 h后,A549细胞增殖和迁移能力降低,差异有统计学意义(P <0.05)。双荧光素酶报告基因法证明PAK4为mi R-433的靶向调节基因,且PAK4在3种NSCLC中的表达均上升。过表达mi R-433后细胞中PAK4、p-LIMK1、p-Cofilin的表达均降低,与mimic control组比较,差异有统计学意义(P <0.05)。结论 mi R-433能通过靶向下调PAK4的表达,抑制LIMK1/Cofilin信号通路,抑制A549细胞的增殖及迁移。

Objective To analyze the effect of miR-433 on the proliferation and migration of A549 cells. Methods The expression of miR-433 in A549, NCI-H1299, NCI-H358 and HFL1 cells was measured by Real-time PCR. A549 cells were transfected with different concentrations an of miR-433 mimic for an optima concentration. Transfection efficiency was measured by Real-time PCR. The cell proliferation was measured by MTT. The cell migration was measured by Transwell. The expression of PAK4, LIMK1, p-LIMK1, cofilin and p-cofilin was measured by western blotting. Results MiR-433 was down-regulated in A549, NCI-H358 compared with HFL1 cells. Overexpression of miR-433 suppressed A549 cell proliferation and migration capability at 48 h (P < 0.05). Dual-luciferase reporter gene assay showed that PAK4 was the target of miR-433. In addition, PAK4 was up-regulated in NSCLCs. Furthermore, overexpression of miR-433 significantly decreased the expression of PAK4, p-LIMK1 and p-cofilin (P < 0.05) compared with those in mimic control group. Conclusion MiR-433 suppresses A549 cell proliferation and migration through inhibiting PAK4/LIMK1/Cofilin pathway.