OBJECTIVE; To determine the effect of a variation of CAG-rich region, which was found 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-beta-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 nuclear 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 transcription activity induced by it may be an important factor to the development T2DM in the carrier.