miR-3168靶向PDCD5调控A549细胞凋亡

miR-3168 targets PDCD5 to regulate apoptosis of A549 cells

目的:探讨miR-3168通过调节PDCD5对非小细胞肺癌A549细胞凋亡的影响。方法:运用TargetScan在线分析miR-3168与PDCD5的相关性;将PDCD5的3′端非编码区克隆进pmiR-RB-Repor Luciferase载体,利用双荧光素酶报告系统检测miR-3168是否靶向调控PDCD5基因;之后用Xfect RNA Transfection Reagent转染miR-3168 mimics或者miR-3168 inhibitor进A549细胞后,用Etoposide处理,通过Western blot检测PDCD5蛋白表达量和A549细胞凋亡标志蛋白的表达量和P53定位情况;同时采用Annexin V染色法测定细胞凋亡水平。结果:通过TargetScan分析,miR-3168仅在一个区域与PDCD5具有较高匹配度;通过双荧光素酶报告系统发现,miR-3168靶向结合PDCD5的3′端非编码区;过表达miR-3168时,PDCD5的表达量下降,细胞凋亡标志蛋白PDCD5的表达量显著下降(P<0.05),细胞凋亡相关蛋白P21和BAK的表达量显著下降(P<0.05),而BCL-2的表达量显著上升(P<0.05),P53入核受到抑制(P<0.05);而用转染miR-3168 inhibitor进A549细胞时,PDCD5的表达量显著上升(P<0.05),细胞凋亡相关蛋白P21和BAK的表达量显著上升(P<0.05),而BCL-2的表达量显著下降(P<0.05),并促进P53入核(P<0.05)。结论:miR-3168通过抑制PDCD5表达量来抑制非小细胞肺癌细胞A549的凋亡。

Objective:To investigate the effect of miR-3168 on the apoptosis of non-small cell lung cancer A549 cells by regulating PDCD5.Methods:The correlation between miR-3168 and PDCD5 was analyzed online using TargetScan.The 3-terminal non-coding region of PDCD5 was cloned into pmiR-RB-Report Luciferase vector,and the dual luciferase reporter system was used to detect whether miR-3168 was targeted PDCD5 gene or not;after transfecting miR-3168 mimics or miR-3168 into A549 cells with Xfect RNA Transfection Reagent,Etoposide treatment was used to detect PDCD5 protein expression and A549 cell apoptosis marker protein expression and P53 localization by Western blot.The situation was also measured by Annexin V staining.Results:By TargetScan analysis,miR-3168 had a high degree of matching with PDCD5 in only one region;miR-3168 was found to bind to the 3-terminal non-coding region of PDCD5 by dual luciferase reporter system;when miR-3168 was overexpressed the expression of PDCD5 decreased,the expression of apoptosis marker protein PDCD5 decreased significantly(P<0.05),the expression of apoptosis-related proteins P21 and BAK decreased significantly(P<0.05),and the expression of BCL-2.The expression of PDCD5 was significantly increased(P<0.05),and the expression of PDCD5 was significantly increased(P<0.05).The expression of BAK and BAK increased significantly(P<0.05),while the expression of BCL-2 decreased significantly(P<0.05),and promoted P53 into the nucleus(P<0.05).Conclusion:miR-3168 inhibits the apoptosis of non-small cell lung cancer cell line A549 by inhibiting the expression of PDCD5.