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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。
Objective To determine the target gene of miR-142-3p in colon cancer SW480 cells and to explore the interaction between miR-142-3p and its target gene. Methods miR-142-3p and negative control sequences were constructed by using miRNA database to predict target genes that might interact with miR-142-3p. MiR-142-3p, control sequence, 3’UTR of TCF7 ) And the mutant TCF7 3’UTR were cloned into the expression vector and co-transfected into SW480 cells. The expression of GFP was detected 36 hours later. The expression of TCF7 mRNA was detected by Trizol RNA extraction and RT-PCR. The expression of TCF7 Protein expression levels. Results Bioinformatics method predicted that TCF7 might be the target gene of miR-142-3p. Further dual luciferase reporter assay confirmed that miR-142-3p could bind TCF7 3’UTR and block TCF7 protein translation, TCF7 protein levels. Conclusion TCF7 may be the target gene of miR-142-3p. MiR-142-3p negatively regulates TCF7 protein expression through transcription.