Genetic variation of rs3811463 is associated with gestational diabetes mellitus susceptibility

Abstract

Gestational diabetes mellitus (GDM) is a growing health concern, and it increases the risk of adverse pregnancy outcomes with substantial long-term adverse health impacts on mothers and their offspring. Several studies have revealed specific associations between genetic variants and the risk of GDM. Single nucleotide polymorphisms (SNPs) are the major type of genetic variation in humans. Let-7 microRNA targets are enriched for genes containing SNPs associated with glucose metabolism, including Lin28. In the present study, the effect of T/C variants of rs3811463 (a SNP located near to the let-7 binding site in Lin28) on GDM risk was investigated. A GDM rat model was successfully constructed using a high fat diet and streptozotocin injection, and the primary skeletal muscle cells were isolated. The cell transfection results demonstrated that rs3811463-T/C significantly affected the glucose uptake and insulin sensitivity. Reverse transcription-quantitative polymerase chain reaction analysis indicated that the C allele at rs3811463 regulated the expression of glucose metabolism-associated genes insulin-like growth factor two binding protein 2 and glucokinase. Western blot analysis data revealed that replacement of the T allele by the C allele at rs3811463 modulated the protein level of Sirtuin 1. Taken together, it was concluded that the let-7/Lin28 axis regulated glucose uptake and insulin sensitivity by modulating the expression of glucose metabolism-associated proteins. These findings provide novel evidence on the association between genetic variations of rs3811463 and GDM susceptibility.

Keywords: Lethal-7; Lin28; gestational diabetes; single nucleotide polymorphism.

Figure 1.

The effect of genetic variations of rs3811463 on glucose uptake and insulin sensitivity. (A) glucose metabolism assay after cell transfection. Muscle cells were pretreated in low-glucose DMEM for 12 h, and then transfected with pEF6/V5-His-TOPO, pEF6/V5-His-TOPO: rs3811463T or pEF6/V5-His-TOPO: rs3811463C, respectively. The radioactivity was determined by scintillation counting. (B) insulin sensitivity analysis after cell transfection. For insulin sensitivity assay, cells were pretreated with 10 µg/ml of insulin for 4 h, and then glucose uptake was performed. All experiments were assayed in triplicate and performed at least four times. Ctrl group, pEF6/V5-His-TOPO-transfected cells; rs3811463T, pEF6/V5-His-TOPO: rs3811463T-transfected cells; rs3811463C, pEF6/V5-His-TOPO: rs3811463C-transfected cells. The letters a and c showed the significant difference (P<0.05) compared with Ctrl group or rs3811463T group, respectively.

Figure 2.

The mRNA levels of let-7 (A), IRS1 (B), IGF2BP2 (C) and GCK (D) after cell transfection determined by qRT-PCR. Muscle cells were pretreated in low-glucose DMEM for 12 h, and then transfected with pEF6/V5-His-TOPO (Ctrl group), pEF6/V5-His-TOPO: rs3811463T (rs3811463T group) or pEF6/V5-His-TOPO: rs3811463C (rs3811463C group), respectively. After 48 h, RNA was isolated for qRT-PCR. GAPDH was used as the internal control. All experiments were performed in triplicate independent repeat analyzed with one-way ANOVA. Compared with Ctrl group, ^ameans P<0.05, ^bmeans P>0.05. Compared with rs3811463T group, ^cP<0.05, ^dP>0.05.

Figure 3.

The relationship between genetic variation of rs3811463 and SIRT1 protein level. Muscle cells were pretreated in low-glucose DMEM for 12 h before 4 h of 10 µg/ml of insulin treatment, and then transfected with pEF6/V5-His-TOPO (Ctrl group), pEF6/V5-His-TOPO: rs3811463T (rs3811463T group) or pEF6/V5-His-TOPO: rs3811463C (rs3811463C group), respectively. Cells were collected and protein was extracted with RIPA buffer for western blot. All experiments were carried out in triplicate independent repeat. All data were analyzed by one-way ANOVA. Compared with Ctrl group, ^aP<0.05; Compared with rs3811463T group, ^cP<0.05.