Role of microRNA 690 in Mediating Angiotensin II Effects on Endoplasmic Reticulum Stress and Inflammation

Abstract

Obesity is a complex disease of global epidemic proportions. Several endocrine systems contribute to obesity, including the renin angiotensin system (RAS), which is overexpressed in adipose tissue during obesity. Overactivation of RAS induces endoplasmic reticulum (ER) stress and inflammation, both hallmarks of obesity; however, mechanisms underlying these interactions are not well known. MicroRNAs (miRNA) plays a pivotal role in post transcriptionally regulating genes involved in obesity and other disorders. Here we propose that changes in RAS alters miRNAs, which thereby regulate ER stress and obesity associated inflammation. This hypothesis was tested using male wild type C57BL/6 mice (Wt) and transgenic mice (Agt‐Tg) overexpressing angiotensinogen (Agt) in the adipose tissue. Mice were fed a low fat (LF) diet and white adipose tissues (WAT) were used to perform microarray and small RNA Seq. High‐throughput computational analyses of differentially expressed genes and miRNAs led to top candidates, which were subsequently validated in WAT. Moreover, specific gene targets for top miRNA candidates were identified using bioinformatic analyses (KEGG pathways, TargetScan). To further validate these miRNA‐gene targets we treated 3T3‐L1 adipocytes with miRNA mimics and inhibitors and tested mRNA expression of target genes. We also mutated the complementary binding sequences on the 3’UTR for positive target genes and validated them in cell culture by dual‐luciferase reporter assay. Here we present data related to miRNA 690 which was significantly altered by Agt overexpression. 23 and 8 miRNAs were upregulated and down regulated respectively in adipose tissue from Agt‐Tg mice. miR690 was significantly upregulated by 3‐fold in Agt‐Tg mice compared to Wt mice and it was also recognized as a potential regulator of several MAPKs including mitogen‐activated protein kinase kinase 3 (MAP2K3). Interestingly, in contrast to other inflammatory genes such as interleukin 6 (IL6) and several MAPKs which were significantly (p<0.05) upregulated in adipose tissue from Agt‐Tg mice compared to Wt mice, MAP2K3 was significantly down regulated in both Agt‐Tg compared to Wt mice and also in 3T3‐L1 cells treated with angiotensin II (Ang II) vs non treated controls. MiRNA 690 mimic treated adipocytes showed significantly reduced Map2k3 expression compared to control. This was further confirmed by significantly reduced luciferase activity for mir690 mimic. Furthermore, mutation of complementary binding sequence of MAP2K3 was unable to reduce luciferase activity, confirming the direct regulatory role of miRNA 690 on MAP2K3. Lastly, a downstream target of MAP2K3, IL‐6 also showed similar results at protein level confirming the involvement of miRNA 690 in regulating inflammation when RAS is overexpressed. In conclusion, miRNA 690 play a protective function with RAS overactivation and miR690 could be used as a potential target to reduce RAS‐induced obesity and related metabolic diseases. Support or Funding Information American Heart Association (NM...