摘要
目的在胃癌细胞中筛选并鉴定靶向Sirt1基因的micorRNA。方法生物信息学软件预测特异性靶向Sirt1基因3’端非编码区域(3′-UTR)的microRNA;分别构建野生型和突变型与目的 microRNA匹配的Sirt1基因3′-UTR序列双荧光素酶报告基因载体;将miR-138-5p过表达质粒分别与野生型和突变型双荧光素酶报告基因载体共转染入胃癌细胞株BGC-823中,应用双荧光素酶检测试剂盒测定荧光素酶活性。miR-138-5p过表达质粒转染BGC-823细胞,荧光定量PCR检测转染细胞miR-138-5p与Sirt1 mRNA的表达,Western blot检测SIRT1蛋白的表达。结果生物信息学软件预测显示miR-138-5p为靶向调控Sirt1的候选micro RNA;成功构建野生型和突变型Sirt1基因3′-UTR双荧光素酶报告基因载体;荧光素酶活性实验表明,miR-138-5p可以下调野生型质粒的荧光素酶活性,不影响突变型质粒的荧光素酶。miR-138-5p过表达质粒转染后的BGC-823细胞中,miR-138-5p表达显著上调,Sirt1基因mRNA和蛋白的表达水平明显下调。结论在胃癌细胞中miR-138-5p靶向作用于Sirt1基因3′-UTR,并负性调控Sirt1基因。
Objective To screen and identify micro RNA of tangeted Sirt1 gene expression in human gastric cancer cells.Methods Bioinformatics software was used to analyze potential micro RNA target sites in 3’-UTR of human Sirt1. The wild type and mutate type of Sirt1 gene 3’-UTR luciferase vectors were constructed respectively. The dual luciferase assay system was used to detect the reporter activity followed by co-transfecting the luciferase vector constructed and miR-138-5p over-expressing plasmid into BGC-823 cells. Furthermore,Sirt1 expressions in m RNA and protein levels following up-regulation of miR-138-5p were detected through q RT-PCR and Western blot. Results Bioinformatics software prediction results showed that miR-138-5p might specifically regulate Sirt1 expression. The wild-type and mutant-type Sirt1 3’-UTR luciferase report vector were constructed successfully. The results of luciferase assays revealed that over expression of miR-138-5p could significantly decrease the luciferase activity of wild type Sirt1 3’-UTR plasmid, not mutant type. The mRNA and protein expression level of Sirt1 were up-regulated in BGC-823 cells when overexpression of miR-138-5p. Conclusion mi-138-5p may negatively regulate the expression of Sirt1 gene in gastric cancer cells by bind to Sirt1 m RNA 3’-UTR directly.
出处
《宁夏医科大学学报》
2016年第9期980-984,993,封4,共7页
Journal of Ningxia Medical University
基金
国家自然科学基金(861260314)
