Objective To investigate the inhibitory effect and mechanism of miR-497-5p on proliferation of human cervical cancer HeLa cells.Methods The human cervical cancer HeLa cells in the logarithmic growth phase were random-ly divided into two parts.The first part was randomly divided into 4 groups:miR-497-5p mimics NC and IKCa1-wt co-transfection group,miR-497-5p mimics and IKCa1-wt co-transfection group,miR-497-5p mimics NC and IKCa1-mut co-transfection group and miR-497-5p mimics and IKCa1-mut co-transfection group which were transfected by corresponding plasmids for 48 h.The luciferase activity of HeLa cells was detected by double luciferase reporter assay system.The second part was randomly divided into 3 groups:miR-497-5p mimics transfection group,miR-497-5p mimics NC transfection group and blank control group.Cells in each group were transfected with Lipofectamin 2000.The expression of IKCa1 mRNA was detected by qRT-PCR,IKCa1 protein expression was detected by Western blotting and the proliferation ability of HeLa cells was measured by CCK-8 assay.Results The luciferase activity of miR-497-5p mimics and IKCa1-wt co-transfection group was lower than that of the other three groups of the first part (all P <0.05),and no significant difference among other three groups (all P >0.05),which confirmed that miR-497-5p and IKCa13 '-URT had specific binding sites.The expression of IKCa1 mRNA and protein of the miR-497-5p mimics transfection group was lower than that of the other two groups of the second part (all P <0.05),and there was no significant difference between the other two groups (all P >0.05).The pro-liferation ability of the miR-497-5p mimics transfection group was lower than that of the other two groups at 24,48,72 and 96 h of culture (all P <0.05),and there was no significant difference between the other two groups (all P >0.05).Con-clusion miR-497-5p can inhibit the proliferation of cervical cancer HeLa cells by negatively regulating the expression of IKCa1 gene.%目的 探讨miR-497-5p对人宫颈癌细胞(HeLa)增殖的抑制作用及机制.方法 将对数生长期人宫颈癌HeLa细胞随机分为两部分,第一部分细胞随机分为四组:miR-497-5p mimics NC+IKCa13′端非翻译区(3′-URT)野生型双荧光素酶重组质粒载体(IKCa1-wt)转染组、miR-497-5p mimics+IKCa1-wt转染组、miR-497-5p mimics NC+3′-URT突变型双荧光素酶重组质粒载体(IKCa1-mut)转染组、miR-497-5p mimics+IKCa1-mut转染组,分别转染相应质粒48 h,用双荧光素酶报告基因检测系统检测HeLa细胞荧光素酶活性.第二部分细胞随机分为三组:miR-497-5p mimics转染组、miR-497-5p mimics NC转染组和空白对照组,分别转染相应质粒,用qRT-PCR技术检测细胞IKCa1 mRNA表达,Western boltting法检测细胞IKCa1蛋白表达,CCK-8法检测细胞增殖能力.结果 第一部分细胞中的miR-497-5p mimics+IKCa1-wt转染组荧光素酶活性较其他三组降低(P均<0.05),其他三组间比较差异无统计学意义(P均>0.05),证实miR-497-5p与IKCa13′-URT存在特异性结合位点.第二部分细胞中的miR-497-5p mimics转染组IKCa1 mRNA、蛋白的相对表达量较其他两组降低(P均<0.05),其他两组比较差异无统计学意义(P均>0.05).第二部分细胞培养24、48、72、96 h,miR-497-5p mimics转染组增殖能力均较其他两组降低(P均<0.05),其他两组比较差异无统计学意义(P均>0.05).结论 miR-497-5p可以通过负向调控IKCa1基因表达以抑制宫颈癌细胞增殖.
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