miR-196a通过靶向NR6A1降低宫颈癌Hela细胞生存和运动能力

miR-196a reduces the survival and mobility of cervical cancer Hela cells via targeting NR6A1

目的探究微小型RNA-196a(miR-196a)对宫颈癌Hela细胞生存和运动能力的影响和作用机制。方法RT-PCR检测正常宫颈上皮HcerEpic细胞和宫颈癌Hela细胞、CaSki细胞中miR-196a和核受体NR6A1的mRNA水平;阴性对照miR-NC和miR-196a inhibitor转染Hela细胞,分别记为NC组和miR-196a inhibitor组,免疫印迹检测NR6A1蛋白的表达,生物信息学和荧光素酶报告实验分析和验证miR-196a和NR6A1的靶向调控关系;阴性对照质粒pcDNA和过表达质粒pcDNA-NR6A1转染Hela细胞,分别记为pcDNA组和pcDNA-NR6A1组,免疫印迹检测NR6A1蛋白表达;将miR-196a inhibitor和pcDNA-NR6A1单独或联合转染Hela细胞,分别记为miR-196a inhibitor组、pcDNA-NR6A1组和inhibitor+NR6A1组,CCK8检测细胞增殖情况,流式细胞术检测细胞凋亡情况,Transwell检测细胞侵袭能力,划痕实验检测细胞迁移能力,免疫印迹检测Ki-67、Caspase-3、VEGF和MMP-9的表达。结果与HcerEpic细胞比较,Hela细胞和CaSki细胞中miR-196a和NR6A1 mRNA表达水平均较高(P<0.01),Hela细胞中miR-196a和NR6A1 mRNA表达变化最为显著(P<0.05);miR-196a inhibitor组Hela细胞中NR6A1蛋白表达显著降低(P<0.01),pcDNA-NR6A1组NR6A1的蛋白表达显著升高(P<0.01);miR-196a inhibitor能显著降低Hela细胞增殖倍数和Ki-67表达水平(P<0.05),同时还能提高Hela细胞凋亡率和Caspase-3表达(P<0.01);此外,miR-196a inhibitor还能减少侵袭细胞数,降低Hela细胞划痕闭合率并抑制VEGF和MMP-9的表达(P<0.01);NR6A1能显著减弱miR-196a inhibitor对Hela细胞增殖、凋亡、侵袭、迁移及对Ki-67、Casapse-3、VEGF和MMP-9表达的调控作用(P<0.01)。结论miR-196a inhibitor能通过靶向抑制NR6A1表达降低宫颈癌Hela细胞的生存和运动能力。

Objective To investigate the effects and mechanism of microRNA-196a(miR-196a)on survival and mobility of cervical cancer Hela cells.Methods The levels of miR-196a and NR6A1 mRNA in normal cervical epithelial HcerEpic cells and cervical cancer Hela and CaSki cells were detected by RT-PCR.Negative control miR-NC and miR-196a inhibitor were transfected into Hela cells,denoted as NC group and miR-196a inhibitor group respectively.The protein level of NR6A1 was detected by Western blot;bioinformatics and luciferase report experiments were used to analyze and verify the targeted regulation relationship between miR-196a and NR6A1.Negative control plasmid pcDNA and overexpression plasmid pcDNA-NR6A1 were transfected into Hela cells,which were denoted as pcDNA group and pcDNA-NR6A1 group respectively,and the expression of NR6A1 was detected by Western blot.The miR-196a inhibitor and pcDNA-NR6A1 were transfected into Hela cells alone or in combination,and the transfected Hela cells were denoted as miR-196a inhibitor group,pcDNA-NR6A1 group and inhibitor+NR6A1 group,respectively.CCK8 assay was performed to detect cell proliferation,flow cytometry was used to detect cell apoptosis,Transwell assay was performed to detect cell invasive ability,wound healing assay was performed to detect cell migration,and Western blot was used to detect the expressions of Ki-67,Caspase-3,VEGF and MMP-9.Results Compared with HcerEpic cells,the expression levels of miR-196a and NR6A1 mRNA in Hela cells and CaSki cells were higher(P<0.01),and the expression of miR-196a and NR6A1 mRNA in Hela cells changed most significantly(P<0.05).The expression of NR6A1 protein in Hela cells in the miR-196a inhibitor group was significantly reduced(P<0.01),while it was significantly increased in pcDNA-NR6A1 group(P<0.01).miR-196a inhibitor significantly decreased the proliferation times and the level of Ki-67(P<0.05),and increased the apoptosis rate of Hela cells and protein level of Caspase-3(P<0.01).In addition,miR-196a inhibitor decreased the number of invasive cells and wound closure rate,and inhibited the expressions of VEGF and MMP-9(P<0.01).NR6A1 significantly alleviated the effects of miR-196a inhibitor on proliferation,apoptosis,invasion,migration and the expressions of Ki-67,Caspase-3,VEGF and MMP-9 in Hela cells(P<0.01).Conclusion miR-196a inhibitor reduces the survival and mobility of cervical cancer Hela cells through targeted inhibiting the expression of NR6A1.