Effect of variation in 5'-regulatory region of insulin receptor substrate-1 gene on gene expression / 中南大学学报(医学版)

ABSTRACT

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.