Functional Characterization of Diabetes‐Induced long non‐coding RNA Dnm3os in macrophages

Abstract

Macrophages play key roles in pathophysiological processes such as inflammation, pathogen response, and tissue repair. Diabetic complications are associated with enhanced macrophage activation and inflammation. Long noncoding RNAs (lncRNAs) regulate gene expression in diverse biological contexts via multiple mechanisms ranging from modulation of chromatin structure and function to post‐transcriptional mechanisms such as mRNA stability. Dysregulation of lncRNA function has been implicated in several diseases, but their role in diabetes complications is unclear. Using high throughput RNA‐sequencing, we recently profiled the transcriptome alterations including lncRNAs in bone marrow derived macrophages from type 2 diabetic (T2D) db/db mice compared to control db/+ mice. In these studies we demonstrated function of a lncRNA in promoting macrophage proinflammatory phenotype. Since the role of other lncRNAs in diabetes complications is largely unknown, we further characterized the function of lncRNA Dynamin 3 opposite strand (Dnm3os), which was upregulated in macrophages from db/db mice. Interestingly, Dnm3os was also increased in high fat diet‐induced insulin‐resistant T2D mice, as well as in monocytes from human T2D patients. Dnm3os was also induced in mouse macrophages treated with diabetogenic agent palmitic acid and proinflammatory agent LPS. RNA‐seq analysis in mouse macrophages stably overexpressing Dnm3os revealed that 167 genes were upregulated and 84 were downregulated compared with control macrophages. DAVID analysis revealed that Dnm3os regulated coding genes were associated with phagocytosis, inflammation and immune response. In contrast, siRNA mediated knockdown of Dnm3os attenuated expression of genes involved in inflammation, immune response and cell activation. RNA‐FISH and RT‐qPCR analysis of subcellular fractions showed that Dnm3os is predominantly localized in the nucleus. Overexpression of Dnm3os altered global histone modification marks in macrophages indicating its role in transcription. In order to determine the molecular mechanisms involved in Dnm3os functions, we performed in vitro RNA pull down assays using biotinylated Dnm3os transcripts combined with mass spectrometry in macrophage nuclear extracts. Results identified that Dnm3os interacts with Nucleolin, ILF2, ARP3 and other RNA binding proteins. RNA‐FISH coupled with Immunofluorescence analyses further confirmed the co‐localization of Dnm3os partially colocalized with nucleolin and ILF2 in the nuclei of macrophages. Collectively, these results demonstrate that Dnm3os has novel functions in diabetic macrophages to promote cellular dysfunction and activation via nuclear mechanisms involving interactions with nucleolin and ILF2. These findings could lead to lncRNA‐based therapies for inflammatory diabetes complications.Support or Funding InformationNIH, NHLBI, NIDDK