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

摘要:

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miR-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miR-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，miR-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miR-433的靶向调节基因，且PAK4在3种NSCLC中的表达均上升。过表达miR-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 miR-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。

Abstract:

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?