【摘要】 目的确定结肠癌SW480细胞中miR-142-3p的靶基因,探讨miR-142-3p与其靶基因的相互作用。方法通过miRNA数据库预测可能与miR-142-3p相互作用的靶基因,构建miR-142-3p及阴性对照序列;将miR-142-3p、对照序列、TCF7的3’非翻译区(3’UTR)以及突变的TCF7 3’UTR分别克隆到表达载体上,共转染SW480细胞,36 h后检测绿色荧光蛋白的表达水平;Trizol抽提RNA,RT-PCR检测TCF7 mRNA表达水平;Western blot检测TCF7蛋白表达水平。结果生物信息学方法预测TCF7可能为miR-142-3p的靶基因,进一步双荧光素酶报告实验验证了miR-142-3p可以与TCF7的3’UTR结合,阻断TCF7蛋白翻译过程,从而下调TCF7的蛋白水平。结论 TCF7可能为miR-142-3p的靶基因,miR-142-3p通过转录后负调控靶基因TCF7蛋白的表达。更多还原

【Abstract】 Objective To determine the target gene of miR-142-3p in colon caner cells SW480 and to investigate the interaction of miR-142-3p with its target gene. Methods The candidate target gene of miR-142-3p was determined according to miRNA analysis databases. The corresponding miR-142-3p and negative control sequence were synthesized. miR-142-3p,control sequence,3’UTR of TCF7 and mutant TCF7 3’UTR were cloned into the expression vectors respectively,and then co-transfected into SW480 cells. After 36 h,the expression level of green fluorescent protein was detected; RNA was extracted by Trizol agent,and RT-PCR was used to detect the expression level of TCF7 mRNA; Western blotting was used to detect the expression level of TCF7 protein. Results Bioinformatic analysis suggested TCF7 might be one of the target genes of miR-142-3p,and further dual luciferase assay indicated that miR-142-3p could combine 3’UTR of TCF7 and block the translation process of TCF7 protein,thus down-regulate the expression level of TCF7 protein. Conclusion TCF7might be the target gene of miR-142-3p,and MiR-142-3p could negatively regulate the expression of TCF7 protein by posttranscription.更多还原