hsa-miR-214-5p通过下调PAK4对子宫颈癌HeLa细胞活力和凋亡的调控作用

Regulatory role of hsa-miR-214-5p on HeLa cells’viability and apoptosis by down-regulating PAK4

目的:探究微小RNA-214-5p(micro RNA-214-5p、miR-214-5p)对宫颈癌HeLa细胞生长和凋亡的作用及机制。方法:将细胞分为HeLa组、mimic-scramble组、miR-214 mimic组和miR-214 inhibitor组,用miR-214 mimic和miR-214 inhibitor转染细胞,qRT-PCR检测miR-214和p21活化激酶4(P21-activated kinases 4, PAK4)的表达;生物信息学方法预测miR-214-5p与PAK4的关系,荧光素酶报告实验验证miR-214-5p与PAK4的关系;用pcDNA-PAK4(pc-PAK4)、miR-214 mimic和miR-214 inhibitor转染细胞,Western blot检测PAK4的表达;CCK-8检测细胞活力;流式细胞术检测细胞凋亡。结果:miR-214 mimic组miR-214-5p mRNA的表达水平(46.02±5.23)较HeLa组(1.00±0.01)明显升高(LSD-t=31.423,P=0.000),miR-214 mimic组PAK4蛋白的表达水平(0.08±0.04)较HeLa组(0.26±0.05)明显降低(LSD-t=4.296,P=0.002);miR-214 inhibitor组miR-214-5p mRNA的表达水平(0.41±0.05)较HeLa组(1.00±0.01)明显降低(LSD-t=18.726,P=0.000),miR-214 inhibitor组PAK4蛋白的表达水平(0.58±0.05)较HeLa组(0.26±0.05)明显升高(LSD-t=5.061,P=0.001);miR-214-5p与PAK4可靶向结合。miR-214 mimic组细胞生长速度(3.50±0.45)较HeLa组(5.02±0.35)明显降低(tD=8.644,P=0.000),miR-214 mimic组细胞凋亡率[(11.42±0.71)%]较HeLa组[(2.61±0.63)%]明显升高(LSD-t=4.032,P=0.003);miR-214 inhibitor组细胞生长速度(5.89±0.32)较HeLa组(5.02±0.35)明显升高(LSD-t=7.863,P=0.000),miR-214 inhibitor组细胞凋亡率[(0.53±0.42)%]较HeLa组[(2.61±0.63)%]明显降低(LSD-t=4.221,P=0.002);共转染pc-PAK4能逆转miR-214 mimic对细胞活力和细胞凋亡的调控作用。结论:miR-214-5p能通过靶向下调PAK4抑制宫颈癌HeLa细胞活力并诱导细胞凋亡。

Objective:To investigate the role of micro-RNA-214-5 p(miR-241-5 p) on the HeLa cells’ cell viability and apoptosis and its mechanism. Methods:HeLa cells were divided into the HeLa group,the mimic-scramble group,the miR-214 mimic group and the miR-214 inhibitor group. The miR-214 mimic and miR-214 inhibitor were used to transfect cells,the qRT-PCR was used to measure expression levels of miR-214 and P21-activated kinases 4(PAK4),the bioinformatic approach was used to predict the relationship of miR-241-5 p and PAK4,and the luciferase reporter assay was used to determine their relationship. The pcDNA-PAK4(pc-PAK4),miR-214 mimic and miR-214 inhibitor were used to transfect HeLa cells,the Western blot was used to determine protein level of PAK4,the CCK-8 assay was used to measure cell viability and the flow cytometry was performed to detect apoptosis. Results:The expression level of miR-214-5 p in the miR-214 mimic group(46.02 ±5.23) was significantly higher than that in the HeLa group(1.00±0.01)(LSD-t=31.423,P=0.000). The expression level of PAK4 in the miR-214 mimic group(0.08±0.04) was significantly lower than that in the HeLa group(0.26 ±0.05)(LSDt=4.296,P=0.002). The expression level of miR-214-5 p in the miR-214 inhibitor group(0.41±0.05) was significantly lower than that in the HeLa group( 1. 00 ± 0. 01)( LSD-t = 18.726,P=0.000). The expression level of PAK4 in the miR-214 inhibitor group(0.58±0.05) was significantly higher than that in the HeLa group of(0.26 ±0.05)(LSD-t=5.061,P=0.001). The miR-214-5 p was able to be targeted-bound with PAK4. The cell growth rate in the miR-214 mimic group(3.50±0.45) was significantly lower than that in the HeLa group(5.02±0.35)(LSD-t=8.644,P=0.000). The apoptosis rate in the miR-214 mimic group [(11.42 ±0.71)% ] was significantly higher than that in the HeLa group[(2.61±0.63)%](LSD-t=4.032,P=0.003). The cell growth rate in the miR-214 inhibitor group(5.89±0.32) was significantly higher than that in the HeLa group(5.02±0.35)(LSD-t=7.863,P=0.000). The apoptosis rate in the miR-214 inhibitor group [(0.53±0.42)%]was significantly lower than that in the HeLa group [(2.61±0.63)%](LSD-t=4.221,P=0.002). Co-transfection of pc-PAK4 was able to reverse the regulation function of miR-214 mimic on cell viability and apoptosis. Conclusion:The miR-214-5 p inhibits the viability of HeLa cells and induces their apoptosis by down-regulating PAK4.