A computational biology approach of a genome-wide screen connected miRNAs to obesity and type 2 diabetes

Abstract

Objective: Obesity and type 2 diabetes (T2D) arise from the interplay between genetic, epigenetic, and environmental factors. The aim of this study was to combine bioinformatics and functional studies to identify miRNAs that contribute to obesity and T2D.

Methods: A computational framework (miR-QTL-Scan) was applied by combining QTL, miRNA prediction, and transcriptomics in order to enhance the power for the discovery of miRNAs as regulative elements. Expression of several miRNAs was analyzed in human adipose tissue and blood cells and miR-31 was manipulated in a human fat cell line.

Results: In 17 partially overlapping QTL for obesity and T2D 170 miRNAs were identified. Four miRNAs (miR-15b, miR-30b, miR-31, miR-744) were recognized in gWAT (gonadal white adipose tissue) and six (miR-491, miR-455, miR-423-5p, miR-132-3p, miR-365-3p, miR-30b) in BAT (brown adipose tissue). To provide direct functional evidence for the achievement of the miR-QTL-Scan, miR-31 located in the obesity QTL Nob6 was experimentally analyzed. Its expression was higher in gWAT of obese and diabetic mice and humans than of lean controls. Accordingly, 10 potential target genes involved in insulin signaling and adipogenesis were suppressed. Manipulation of miR-31 in human SGBS adipocytes affected the expression of GLUT4, PPARγ, IRS1, and ACACA. In human peripheral blood mononuclear cells (PBMC) miR-15b levels were correlated to baseline blood glucose concentrations and might be an indicator for diabetes.

Conclusion: Thus, miR-QTL-Scan allowed the identification of novel miRNAs relevant for obesity and T2D.

Keywords: Adipogenesis; Computational biology; Insulin signalling; QTL; Type 2 diabetes; miR-31.

Figure 1

Workflow of the miR-QTL-Scan for the identification of miRNAs and the putative targets genes in obesity- and diabetes-related QTL. Numbers refer to working steps. Step III is optional and used to identify tissue-specific miRNAs. gWAT: gonadal white adipose tissue.

Figure 2

Overview of miRNAs located in QTL for obesity and T2D and differently expressed target genes. Circos plot of miRNAs located in QTL for obesity (A) and T2D (B), predicted targets, targets in experimental databases, and putative differentially expressed targets in gWAT, BAT, and muscle. (C) Number of differentially expressed genes (DEG) in BAT, WAT, and skeletal muscle of B6 and NZO mice. gWAT: gonadal white adipose tissue; scWAT: subcutaneous white adipose tissue; BAT: brown adipose tissue; DEG: differently expressed genes.

Figure 2

Overview of miRNAs located in QTL for obesity and T2D and differently expressed target genes. Circos plot of miRNAs located in QTL for obesity (A) and T2D (B), predicted targets, targets in experimental databases, and putative differentially expressed targets in gWAT, BAT, and muscle. (C) Number of differentially expressed genes (DEG) in BAT, WAT, and skeletal muscle of B6 and NZO mice. gWAT: gonadal white adipose tissue; scWAT: subcutaneous white adipose tissue; BAT: brown adipose tissue; DEG: differently expressed genes.

Figure 3

Position of miR-31 in the obesity QTL Nob6 on chromosome 4, the predicted and validated targets and differentially expressed genes in gWAT of B6 and NZO mice. (A) Peak regions of the QTL for body weight (BW) in week 6/14 and weight of gWAT. Peak of LOD-score is added to the bottom axis. (B) Comparison of differently expressed genes in gWAT with predicted targets and experimentally validated targets of miR-31. (C) Pathway enrichment analysis for the 416 target genes of miR-31 in gWAT, filtered for a fold enrichment >3 and p < 0.1. (D) Heat map for a two-dimensional genome scan with a two-QTL model for the NZOxB6 cross. The maximum LOD score for the interaction model (locus to locus interaction) in the upper left triangle. The maximum LOD score for the full model (two QTLs plus additive effect) is indicated in the lower right triangle. Black squares show an interaction between miR-31 and predicted genes (Foxo1, Hk2, Irs1, Glut4, Acaca), LOD-score >6 (dark red). gWAT: gonadal white adipose tissue.

Figure 4

Expression of miR-31 target genes in gWAT and tissue-specific expression pattern of miR-31 in B6 and NZO mice. Differential expression of (A) miR-31-5p and (B) miR-31-3p target genes involved in insulin signaling pathway in gWAT detected by array analysis. (n = 4/group). (C) Expression of miR-31-5p and miR-31-3p in gWAT and (D) in liver of 6 weeks old B6 (n = 5) and NZO (n = 6) mice. (*p < 0.05, **p < 0.001, ***p < 0.0001). Data are presented as ± SEM. gWAT: gonadal white adipose tissue. (E) Partial sequences of the putative targets (human in magenta, mouse in black, miRNA in blue) of miR-31-5p and (F) of miR-31-3p. The seed regions of the targets are indicated as red dashes.

Figure 4

Expression of miR-31 target genes in gWAT and tissue-specific expression pattern of miR-31 in B6 and NZO mice. Differential expression of (A) miR-31-5p and (B) miR-31-3p target genes involved in insulin signaling pathway in gWAT detected by array analysis. (n = 4/group). (C) Expression of miR-31-5p and miR-31-3p in gWAT and (D) in liver of 6 weeks old B6 (n = 5) and NZO (n = 6) mice. (*p < 0.05, **p < 0.001, ***p < 0.0001). Data are presented as ± SEM. gWAT: gonadal white adipose tissue. (E) Partial sequences of the putative targets (human in magenta, mouse in black, miRNA in blue) of miR-31-5p and (F) of miR-31-3p. The seed regions of the targets are indicated as red dashes.

Figure 5

Expression of miR-31-5p and miR-31-3p in human adipose tissue. (A) miR-31 sequence similarity between humans and mice. (B) miR-31-5p and miR-31-3p expression levels in visceral and (C) subcutaneous adipose tissue of healthy (n = 15–17), obese (n = 34–37), and diabetic (T2D, n = 33–35) subjects analyzed by qRT-PCR. Obese subjects had a BMI >30. (*p < 0.05, ***p < 0.0001). Data are presented as ± SEM.

Figure 6

Impact of miR-31 mimic during differentiation in human adipocytes. (A) Oil red O staining of adipocytes in the culture dish of SGBS cells at the indicated days after induction of differentiation. (B) Percentage of differentiated adipocytes and glycerol levels during differentiation. (C) Expression levels of miR-31-5p and miR-31-3p in SGBS cells at indicated days after induction of differentiation (n = 3). (D) Expression of miR-31-5p at the indicated time points of differentiation after transfection with a miR-31-5p-specific mimic 2 days before treating SGBS cells with the differentiation cocktail. (E) mRNA expression of putative miR-31-5p targets at the indicated time points after differentiation. (*p < 0.05, **p < 0.01) Data are presented as ± SEM.

Figure 7

Analysis of miRNAs in human PBMCs. (A) Correlation of miR-15b-5p expression with fasted blood glucose levels (n = 89). (B) Expression of miR-15b, miR-31, miR-744 and miR-30b in PBMCs of healthy (NGT; BMI < 30; n = 25), obese (NGT; BMI > 30; n = 16), diabetic (IGT; BMI < 30; n = 20) and diabetic and obese (IGT; BMI > 30; n = 28) patients. Data are presented as ± SEM.