微小RNA-106b靶向下调腺瘤性息肉病基因表达促进Hep-2细胞的增殖

摘要

Objective To explore the molecular mechanism of microRNA (miRNA,miR)-106b on laryngeal squamous carcinoma Hep-2 cells proliferation capacity.Methods Methyl thiazol tetrazolium(MTT) assay was used to detect the influence of miR-106b on Hep-2 cell proliferation.Real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting were used to demonstrate the expression of adenomatous polyposis coli (APC) mRNA and protein respectively after transfection of Hep-2 cells with miR-106b-mimics,control miR and miR-106b inhibitor.The dual luciferase assay system was used to detect the reporter activity followed by co-transfecting the luciferase vector construted and miR-106b-mimics,control miR and miR-106b inhibitor in Hep-2 cells.Results miR-106b-mimics transfection can significantly increase the proliferation capacity of Hep-2 cell line compared to control miR group and miR-106b inhibitor group.In those three group,APC protein level was 0.586 ±0.023,0.734 ± 0.024 and 0.927 ± 0.018 respectively,which has a significantly difference (P ＜ 0.05),but no effect on the level of APC mRNA (P ＞ 0.05).miR-106b-mimics group,control miR group and miR-106b inhibitor group were constructed,the luciferase assay revealed that the luciferase activity of wide type APC 3' untranslated region (3' UTR) vector was 4.37 ± 1.04,8.376 ± 1.23 and 23.75 ± 1.63 respectively,which has a significantly difference in three groups (P ＜ 0.05),but no effect on the mutation type APC 3' UTR vector of luciferase activity (P ＞ 0.05).Conclusion miR-106b could negatively regulate the expression of APC by binding to the 3' UTR,which lead laryngeal squamous carcinoma Hep-2 cell proliferation capacity increased.%目的 探讨微小RNA(miRNA,miR)-106b影响喉鳞状细胞癌Hep-2细胞株增殖的分子机制.方法 应用噻唑蓝(MTT)法分析miR-106b对Hep-2细胞株增殖的影响;通过实时荧光定量聚合酶链反应(FQ-PCR)和Western blot技术检测miR-106b各转染组腺瘤性息肉病基因(APC) mRNA和蛋白表达水平;将APC基因3'非翻译区(3'UTR)-荧光素酶表达载体及其突变体与miR-106b共转染至Hep-2细胞,采用双荧光素酶检测系统检测荧光素酶的活性.结果 与miR-106b inhibitor和对照组比较,转染miR-106b-mimics能明显促进Hep-2细胞的增殖;APC蛋白在miR-106b-mimics转染组、对照组和miR-106b inhibitor中的表达水平分别为0.586 ±0.023,0.734 ±0.024和0.927±0.018,3组间差异有统计学意义(P＜0.05);而APC mRNA的表达水平在3组间差异无统计学意义(P＞0.05);在共转染野生型APC基因3'UTR-荧光素酶表达载体的Hep-2细胞中,上述3组荧光素酶的活性分别为4.37±1.04,8.376±1.23和23.75±1.63,3组间差异有统计学意义(P＜0.05);而在共转染突变型APC基因3'UTR-荧光素酶表达载体的Hep-2细胞中,上述3组荧光素酶的活性差异无统计学意义(P＞0.05).结论 在喉鳞状细胞癌Hep-2细胞株中,miR-106b可结合到APC基因3'UTR,在转录后水平负性调控APC基因的表达,从而促进Hep-2细胞的增殖.