胰岛素受体底物-1基因5'-调控区的一种变异对基因表达的影响

摘要

Objective To determine the effect of a variation of CAG-rich region,which was fond in the 5'-regulatory sequence of insulin receptor substrate-1(IRS-1)gene in Type 2 diabetes mellitus(T2DM)patients,on gene expression and its mechanism.Methods The recombinants,pGL2.P-T3 and pGL2.P-T5,were constructed with luciferase reporter vector,pGL2 promoter.T3 and T5 were wild-type and variant alleles,respectively.The recombinants were cotransfected with pSV-β-galactosidase control vector to Hela cells.Luciferase assay was performed to assess transcriptional actiVity.The electrophoresis mobility shift assay(EMSA)and DNA footprint assay were applied to determine the interaction between the DNA regulatory sequences and nuclear proteins of Hela cells.Results The relative transcription activity of T5 was lower than that of T3[(7.76±1.05)%vs(9.98±1.40)%,P＜0.05];EMSA showed both T3 and T5 formed a single retarded band in gel with the same mobility with nnclear proteins;T5 had 2 binding sites for transacting factors,CGCGCCCGCGGGCGGCGGC and GGGCGGCTGGTGGCGGCTG,which was the same as T3.Conclusion Although the variation in T5 do not alter the DNA-binding sites for Hela cell nuclear extracts,the notable decrease in gene transcrip tionactivity induced by it may be an important factor to the development T2DM in the carrier.%目的:研究2型糖尿病患者胰岛素受体底物-1(IRS-1)基因5'-调控区CAG富含区的一种变异对基因表达活性的影响及其机制.方法:构建荧光素酶(luciferase,luc)报告基因重组体:pGL2.P-T3和pGL2.P-T5,T3和T5分别为野生型和缺失型等位基因.重组体与pSV-β-Galactosidase Control Vector共转染Hela细胞,通过luc活性分析检测基因的转录活性;采用凝胶滞留和DNA足纹技术,确定DNA调节序列和Hela细胞核蛋白的相互作用.结果:T5的相对活性低于T3[(9.98±1.40)%和(7.76±1.05)%,P＜0.05];凝胶滞留实验表明与核蛋白作用后,T3和T5在凝胶上形成一条迁移率相同的滞留区带;T5有2个反式作用因子结合位点,CGCGCCCGCGGGCGGCGGC和GGGCGGCTGGTGGCGGCTG,与T3相同.结论:尽管T5的变异未改变Hela细胞核蛋白提取物的DNA结合位点,但是它引起的转录活性的下降可能是携带者2型糖尿病发生发展的重要因素.