miR-499 rs3746444多态性抑制宫颈癌细胞的增殖作用及分子机制

目的探讨miR-499 rs3746444多态性与宫颈癌临床特征相关性及对宫颈癌细胞增殖的影响作用与机制。方法应用病例对照研究方法,选取2012年6月至2018年12月于内蒙古自治区人民医院就诊的宫颈癌病人857例作为实验组,同期873例健康志愿者作为对照组,两组年龄均在23~62岁,采用TaqMan探针法对rs3746444位点的多态性进行基因分型;构建包含pGL3-Sox63'UTR和miR-499的GG或AA的海肾荧光素酶载体(Renilla luciferase vector)于prl-sV40质粒中,并采用lipo2000转染人宫颈上皮永生化细胞H8和人宫颈癌细胞系:腺癌样SiHa、上皮样HeLa和C33A,CCK-8法检测细胞增殖;双荧光素报告基因检测法分析Frefly和Renilla荧光素酶活性;Western blot检测Hela和SiHa细胞Cyclin D1、CyclinE、CDK4、CDK6和Sox6蛋白表达。结果AG和GG基因型宫颈癌发生风险分别是AA基因型个体的1.09倍和2.41倍,携带G等位基因的个体患宫颈癌的风险是AA基因型个体的1.28倍,GG基因型宫颈癌患者III期及以上的比例显著高于AA基因型。GG表型肿瘤高度分化比例明显高于AA表型组,rs3746444不同基因型对正常宫颈H8细胞的增殖无显著影响。miR-499 GG+Sox6-3'UTR处理组Hela、C33A和SiHa细胞72 h时OD450显著低于miR-499 AA+Sox6-3'UTR组,且高于Sox6-3'UTR组(P<0.05)。miR-499 GG+Sox6-3'UTR处理组Hela和SiHa细胞Cyclin D1、CyclinE、CDK4和CDK6显著高于miR-499 AA+Sox6-3'UTR组,低于Sox6-3'UTR组(P<0.05)。与miR-499 AA+Sox6-3'UTR处理组相比,miR-499 GG+Sox6-3'UTR处理组Sox6的表达水平增加(P<0.05),且均低于Sox6-3'UTR对照组(P<0.05)。结论miR-499的rs3746444突变(A>G)与宫颈癌肿瘤生长和分化程度密切相关,其通过上调Sox6介导的CyclinD1高表达具有明显的促进宫颈癌细胞生长增殖作用。

Inhibitory effect of miR-499 rs3746444 polymorphism on proliferation of cervical cancer cells and its molecular mechanism

Objective To investigate the correlation between miR-499 rs3746444 polymorphism and clinical features of cervical cancer,and its effect on the proliferation of cervical cancer cells.Methods Case-control study was conducted among 857 patients with cervical cancer from June 2012 to December 2018 in the Inner Mongolia people's hospital,and 873 healthy volunteers were included as the controls.The age of both groups were 23~62 years old.The polymorphism of miR-499 at rs3746444 was genotyped by TaqMan probe method.And the pGL3-Sox6 was constructed Renilla luciferase vector of GG or AA of 3'UTR and miR-499 was inserted into prl-sv40 plasmid.Lipo2000 was used to transfect human cervical epithelial immortalized cells H8 and human cervical cancer cell lines:adenocarcinoma like SiHa,epithelioid HeLa and c33a.Cell proliferation was detected by CCK-8 assay;The luciferase activities of frefly and Renilla were analyzed by double luciferase reporter gene assay,and the protein expressions of cyclin D1,cyclinE,CDK4,Cdk6 and Sox6 in HeLa and SiHa cells were detected by Western blot.Results The risk of cervical cancer in the AG and GG genotypes was 1.09 and 2.41 times higher than that of the AA genotype,respectively.The risk of cervical cancer in individuals carrying the G allele was 1.28 times that of the AA genotype.The proportion of patients at stage III and high differentiation features in cervical cancer patients with GG genotype was significantly higher than that of AA genotype.The different genotypes of rs3746444 had no significant effect on the proliferation of normal cervical H8 cells.The OD 450 of Hela,C33A and SiHa cells treated with miR-499 GG+Sox6-3'UTR was significantly lower than that of miR-499 AA+Sox6-3'UTR group at 72h,and higher than that of Sox6-3'UTR group(P<0.05).The Cyclin D1,CyclinE,CDK4 and CDK6 in HeLa and SiHa cells of miR-499 GG+Sox6-3'UTR treatment group were significantly higher than those of miR-499 AA+Sox6-3'UTR group,lower than Sox6-3'UTR group(P<0.05).Compared with the miR-499 AA+Sox6-3'UTR-treated group,the expression level of Sox6 in the miR-499 GG+Sox6-3'UTR-treated group was increased(P<0.05).Both were lower than the Sox6-3'UTR control group(P<0.05).Conclusion The rs3746444 mutation(A>G)of miR-499 is closely related to the growth and differentiation of cervical cancer.It can significantly promote the growth and proliferation of cervical cancer cells by up-regulating the overexpression of CyclinD1 mediated by Sox6.