miR-497-5p对人宫颈癌细胞增殖的抑制作用及机制

Inhibitory effect of miR-497-5p on proliferation of human cervical carcinoma cell

目的探讨miR-497-5p对人宫颈癌细胞(He La)增殖的抑制作用及机制。方法将对数生长期人宫颈癌He La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+IKCa1 3'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He La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IKCa1 3'-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。

Objective To investigate the inhibitory effect and mechanism of miR-497-5p on proliferation of human cervical cancer He La cells. Methods The human cervical cancer He La cells in the logarithmic growth phase were randomly divided into two parts. The first part was randomly divided into 4 groups： miR-497-5p mimics NC and IKCa1-wt cotransfection group,miR-497-5p mimics and IKCa1-wt co-transfection group,miR-497-5p mimics NC and IKCa1-mut cotransfection 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 He La 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 He La 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 IKCa1 3 ＇-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 proliferation ability of the miR-497-5p mimics transfection group was lower than that of the other two groups at 24,48,72 and96 h of culture（ all P 0. 05）,and there was no significant difference between the other two groups（ all P 0. 05）. Conclusion miR-497-5p can inhibit the proliferation of cervical cancer He La cells by negatively regulating the expression of IKCa1 gene.