The role of altered miRNA expression in a variety of diseases is increasingly being recognized but the precise nature and downstream effects of such changes is largely unknown. Previous studies have shown that miRNAs regulate various physiological events relevant to T2D pathophysiology, such as insulin secretion, insulin responsiveness and energy homeostasis. Here, we have characterized differential miRNA and mRNA expression in insulin sensitive tissues of rats of a model of T2D (GK rat) and in normoglycaemic BN controls, a strain combination extensively used to study the genetic determinants of T2D phenotypes in F2 hybrids and congenic strains . In the miRNA data, the most striking finding was the over-expression of miR-125a in both liver and adipose tissue (with nearly 6-fold and 2-fold higher expression, respectively) in GK compared to BN rats. This finding was validated by RT-PCR in liver (FC = 13.15, p = 0.0005). The higher sensitivity and dynamic range of the RT-PCR technique is the most likely reason why the fold change is higher than detected by microarray.
MiR-125a and its close homolog miR-125b differ by a single nucleotide , and thus share many predicted target genes. Experimentally validated target genes of both miR-125a and miR-125b in humans include ERBB2 and ERBB3  and LIN28 . Both miR-125a and miR-125b have been reported to be down-regulated in ovarian  and breast cancers , with potential roles in cell proliferation and differentiation. To further investigate the potential role of miR-125a in T2D, we assessed the functional roles of predicted miR-125a target genes. This analysis suggested that increased miR-125a levels may particularly affect genes involved in the MAPK signalling pathway. MAPK has been implicated in T2D [28, 30] and plays a critical role in insulin signalling , and may contribute to insulin resistance . Further functional experiments will help elucidate the role of MAPK signalling in T2D and to what extent increased miR-125a expression may affect this pathway.
We integrated our miRNA results with gene expression data from the same animals and found an enrichment of miR-125a target genes predicted by the miRanda algorithm  among genes down-regulated in GK rats in adipose tissue. Of particular interest, a proposed functional candidate gene for T2D, Ptges2, is a predicted miR-125a target gene and was significantly down-regulated in both tissues. Two other miR-125a target genes of interest in the context of T2D and obesity are Ppap2c and Sult1a1, both of which were significantly down-regulated in adipose tissue from the GK rat. In addition; single nucleotide polymorphisms (SNPs) in the SULT1A1 region have previously been associated with increased BMI in humans . Contrary to expectation, it was surprising to find an even stronger enrichment among the up-regulated genes. One possibility is that these are not direct target genes themselves, but represent changes further downstream of the miRNA regulation. No enrichment was observed with the target gene lists predicted by the other two algorithms, which may be due to differences in the target-gene prediction methods implemented or to the effects of long-term exposure to hyperglycaemia further affecting the control of gene expression. These results show that the relationship between miRNAs and gene expression is not a simple one.
Though the enrichment of miR-125a target genes predicted by miRanda is an interesting finding, it raises a number of questions, including why both down and up-regulated genes should show significant enrichment of miR-125a target genes, and why stronger enrichment was observed for the tissue with lower fold induction of miR-125a. The difficulty of predicting genuine target genes combined with other influences on gene expression could provide some explanation. Furthermore, the current data do not demonstrate whether the expression of any of the genes is dysregulated as a direct consequence of the increased miR-125a levels. One approach to identify genes likely to be regulated by miR-125a is to find those that show a strong negative correlation between their expression and miR-125a levels. Unfortunately, the two-colour experimental design used for the miRNA study (generating GK v BN expression ratios) combined with the small sample size precluded such an analysis here. Integrating miRNA and mRNA data has the potential to give further insights into miRNA-mediated regulation of gene expression but is limited to detecting events in which the target mRNA is degraded. Still, in-silico approaches will likely be useful for prioritizing target genes for functional validation.
Our results suggest that increased miR-125a expression may be a characteristic feature of hyperglycaemic GK rats. However, as this study was carried out in rats with long-established T2D phenotypes, it remains unclear whether the observed changes are causative, or reflect adaptation to prolonged hyperglycaemia. GK and BN rats are also genetically different strains, and it is therefore possible that strain differences unrelated to hyperglycaemia could contribute to the altered expression of miR-125a. Although in-silico functional profiling of gene expression data and miR-125a target genes lends some support to the idea that miR-125a over-expression is linked to the hyperglycaemic phenotype, further evidence from other model systems or functional studies is certainly desirable.
Among the other miRNAs showing robust differences between GK and BN rats was a 1.5 fold up-regulation of miR-29a in adipose tissue. This is exactly comparable with the only previous study of miRNAs in T2D, which identified a 1.5 fold up-regulation of miR-29a, together with the paralogs 29b and 29c, in skeletal muscle from GK and Wistar rats (normoglycaemic controls). Northern blot analysis confirmed the up-regulation of all three miR-29 paralogs in muscle, adipose tissue and liver. This study further showed that high glucose and insulin (which indicate insulin resistance) up-regulate the expression of miR-29a/b in adipocyte cell lines. They also suggest the over-expression of miR-29a/b inhibits insulin-induced glucose import in the same adipocyte cell lines. Taken together, these data support the involvement of miR-29a in insulin resistance and the insulin-signaling pathway.