miR-224-5p靶向调控KIF23通过NF-κB信号通路影响宫颈癌细胞增殖、迁移和侵袭

miR-224-5p targeted regulation KIF23 and affected the proliferation,migration and invasion of cervical cancer cells through the NF-κB signaling pathway

目的:探索微小RNA-224-5p(miR-224-5p)对驱动蛋白超家族23(KIF23)的靶向关系及对宫颈癌细胞增殖、迁移和侵袭的影响。方法:采用实时定量PCR(qPCR)检测正常宫颈细胞(H8)和宫颈癌细胞(SiHa、HeLa、MS751、HT-3)中miR-224-5p和KIF23 mRNA表达,选择miR-224-5p表达量最低的HeLa细胞开展后续研究。在HeLa细胞中转染si-KIF23或miR-224-5p,探讨敲减KIF23或过表达miR-224-5p对HeLa细胞增殖、迁移和侵袭的影响。噻唑蓝(MTT)比色法检测细胞增殖,Transwell小室法检测细胞迁移和侵袭,蛋白质印迹法(Western Blot)检测KIF23、细胞周期蛋白D1(CyclinD1)、基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)和NF-κBp65蛋白水平;生物学信息预测和双荧光素酶报告基因实验分析miR-224-5p和KIF23之间的靶向关系;共转染si-KIF23和anti-miR-224-5p,观察miR-224-5p低表达对敲减KIF23诱导的HeLa细胞增殖、迁移、侵袭和NF-κB信号通路活化的影响。结果:miR-224-5p在SiHa、HeLa、MS751、HT-3细胞中表达下调(P<0.05),KIF23 mRNA和蛋白表达上调(P<0.05)。敲减KIF23或过表达miR-224-5p显著降低HeLa细胞存活率、细胞迁移数和侵袭数,并显著抑制CyclinD1、MMP2、MMP9蛋白水平(P<0.05)。另外,敲减KIF23显著降低细胞中NF-κBp65表达量(P<0.05)。miR-224-5p靶向调控KIF23表达。miR-224-5p低表达可以逆转敲减KIF23抑制HeLa增殖、迁移、侵袭及CyclinD1、MMP2、MMP9蛋白表达的作用,以及逆转敲减KIF23抑制NF-κB信号通路活化的作用。结论:miR-224-5p靶向调控KIF23并通过NF-κB信号通路抑制宫颈癌细胞增殖、迁移和侵袭。

Objective:To investigate the targeting relationship of microRNA-224-5 p(miR-224-5 p)on Kinesin family member 23(KIF23)and the effects on proliferation,migration and invasion of cervical cancer cells.Methods:Real-time quantitative PCR(qPCR)was used to detect the expression of miR-224-5 p and KIF23 in normal cervical cells(H8)and cervical cancer cells(SiHa,HeLa,MS751,HT-3).The HeLa cells with the lowest expression of miR-224-5 p were selected for further research.The effects of knocking down KIF23 or over-expressing miR-224-5 p on the proliferation,migration and invasion of HeLa cells were studied by transfecting si-KIF23 or miR-224-5 p into HeLa cells.MTT colorimetric assay was used to determined cell proliferation.Transwell cell assay was applied to detect cell migration and invasion.Western Blot was employed to inspect the levels of KIF23,CyclinD1,Matrix metalloproteinase 2(MMP2),Matrix metalloproteinase 9(MMP9)and NF-κBp65.Bioinformatics prediction and dual luciferase reporter gene assay were used to confirm the targeting relationship between miR-224-5 p and KIF23.si-KIF23 and anti-miR-224-5 p were co-transfected to observe the effects of low expression of miR-224-5 p on cell proliferation,migration,invasion and activation of NF-κB signaling pathway induced by knocking down KIF23.Results:The expression of miR-224-5 p was down-regulated in SiHa,HeLa,MS751 and HT-3 cells(P<0.05),and the expression of KIF23 was up-regulated(P<0.05).Knockdown of KIF23 or over-expression of miR-224-5 p obviously reduced the survival rate,cell migration and invasion of HeLa cells,and significantly inhibited the levels of CyclinD1,MMP2 and MMP9 proteins(P<0.05).In addition,knockdown of KIF23 significantly decreased the expression of NF-κBp65(P<0.05).miR-224-5 p targeted regulation the expression of KIF23.The low expression of miR-224-5 p reversed the inhibition of KIF23 on HeL a proliferation,migration,invasion and the expression of CyclinD 1,MMP2 and MMP9 proteins,as well as the inhibition of KIF23 on the activation of NF-κB signaling pathway.Conclusion:miR-224-5 p targets KIF23 and inhibits the proliferation,migration and invasion of cervical cancer cells through NF-κB signaling pathway.