Integrative mRNA-microRNA analyses reveal novel interactions related to insulin sensitivity in human adipose tissue

Abstract

Adipose tissue has profound effects on whole-body insulin sensitivity. However, the underlying biological processes are quite complex and likely multifactorial. For instance, the adipose transcriptome is posttranscriptionally modulated by microRNAs, but the relationship between microRNAs and insulin sensitivity in humans remains to be determined. To this end, we utilized an integrative mRNA-microRNA microarray approach to identify putative molecular interactions that regulate the transcriptome in subcutaneous adipose tissue of insulin-sensitive (IS) and insulin-resistant (IR) individuals. Using the NanoString nCounter Human v1 microRNA Expression Assay, we show that 17 microRNAs are differentially expressed in IR vs. IS. Of these, 16 microRNAs (94%) are downregulated in IR vs. IS, including miR-26b, miR-30b, and miR-145. Using Agilent Human Whole Genome arrays, we identified genes that were predicted targets of miR-26b, miR-30b, and miR-145 and were upregulated in IR subjects. This analysis produced ADAM22, MYO5A, LOX, and GM2A as predicted gene targets of these microRNAs. We then validated that miR-145 and miR-30b regulate these mRNAs in differentiated human adipose stem cells. We suggest that use of bioinformatic integration of mRNA and microRNA arrays yields verifiable mRNA-microRNA pairs that are associated with insulin resistance and can be validated in vitro.

Keywords: adipose; insulin sensitivity; microRNA; microarray.

Fig. 1.

Bioinformatic analyses schematic. Differentially expressed mRNA and microRNAs were identified in adipose tissue of insulin-sensitive (IS) and insulin-resistant (IR) individuals. Potential biological interaction were identified from reciprocal expression patterns, followed by inverse correlations. Gene regulation by specific microRNAs was confirmed in vitro.

Fig. 2.

Hierarchical clustering of differentially expressed microRNAs based on insulin sensitivity (S_I). Hierarchical clustering reveals discrete clustering of microRNA profiles based on an individual's insulin sensitivity. Sixteen out of 17 differentially expressed microRNAs are lower in IR vs. IS subjects.

Fig. 3.

microRNA (miR)-145 (A), -26b (B), and -30b (C) demonstrate strong positive relationships with insulin sensitivity. MicroRNA expression was determined by NanoString technology and correlated against individuals Log S_I. P < 0.01.

Fig. 4.

miR-145 and miR-30b regulate the expression of their predicted mRNA targets. ADHASC cells from 3 different individuals were transfected with miR-145, miR-30b, or scrambled control. miR-145: MYO5A (A), GM2A (B), LOX (C), ADAM22 (D); miR-30b: ADAM22 (E). Data are presented as percent reduction in gene expression within each different cell line. *P < 0.05.

Fig. 5.

MYO5A, LOX, GM2A, and ADAM22 expression levels are negatively associated with insulin sensitivity. Gene expression was determined by Agilent microarray and correlated against individuals Log S_I. A: MYO5A; B: GM2A; C: LOX; D: ADAM22. P < 0.0001