Polymorphism in microRNA-binding site in HNF1B influences the susceptibility of type 2 diabetes mellitus: a population based case-control study

Abstract

Background: Recent genome-wide association studies (GWAS) have identified many SNPs associated with type 2 diabetes mellitus (T2DM). However, the functional roles for most of the SNPs have not been elucidated. MicroRNAs (miRNAs) are key regulators of gene expression involved in the development and progression of various diseases including T2DM. In this study, we investigated whether commonly occurring SNPs modulate miRNA-directed regulation of gene expression, and whether such SNPs in miRNA-binding sites are associated with the susceptibility for T2DM.

Methods: Genotypes of eleven 3' untranslated region (UTR) SNPs of seven susceptibility genes for T2DM were determined in 353 T2DM patients and 448 control subjects. In addition, the interactions of miRNAs with the 3'UTR in the hepatocyte nuclear factor 1β (HNF1B) gene were investigated using luciferase reporter assays.

Results: One 3'UTR SNP (rs2229295) in the HNF1B gene was significantly associated with T2DM, and the frequency of an A allele (rs2229295) in T2DM patients was decreased compared with that in controls. Luciferase reporter assays showed that the SNP (rs2229295) altered the binding of two miRNAs (hsa-miR-214-5p and hsa-miR-550a-5p).

Conclusions: We have detected the interactions of hsa-miR-214-5p/hsa-miR-550a-5p and the 3'UTR SNP of the HNF1B gene by in vitro luciferase reporter assays, and propose that the binding of such miRNAs regulates the expression of the HNF1B gene and the susceptibility of T2DM.

Fig. 1

Predicted miRNAs whose binding are possibly affected by the base substitutions due to SNPs r22229295 and rs1800929. The four miRNAs were predicted as candidate miRNAs in at least two of three online databases (MirSNP, PolymiRTS, and miRNASNP) [–25]. Seed sequences of each miRNA were indicated by bold. The complemetary sequences of 3′UTR of the HNF1B gene were shown by underlined. The red color showed sites for SNPs (rs2229295 and rs1800929)

Fig. 2

Effect of the base substitutions due to SNPs rs2229295 and rs1800929 on miRNA binding. a Schematic representation of reporter constructs used in the luciferase reporter assay. Plasmid construct containing TC sequence, which was selected randomly, was used as a reference. Major allele (C for rs2229295) is shown in blue and minor allele (A for rs2229295) is shown in red. b Relative luciferase activity of each reporter construct. Luciferase activity was normalized to Renilla luciferase levels. Luciferase activities relative to the reference vector (TC vector) are shown as mean ± S.E. from three independent transfection experiments with triplicate assays. The luciferase activities among four constructs were compared using the Turkey-Kramer method (*p < 0.05, **p < 0.01)