MiR-449a regulates insulin signalling by targeting the Notch ligand, Jag1 in skeletal muscle cells

Shagun Poddar,Devesh Kesharwani,Malabika Datta

doi:10.1186/s12964-019-0394-7

Abstract

BackgroundmiR-449a, an intronic miRNA, is highly down-regulated in the skeletal muscle during diabetes. Its levels are epigenetically regulated by altered acetylation/deacetylation on the promoter that it shares with its host gene, Cdc20b. However, the cellular role of this epigenetically regulated miRNA in the muscle during diabetes is not well understood. Here, we sought to unravel the crosstalk between altered miR-449a expression and impaired skeletal muscle metabolism.MethodsPredicted targets of miR-449a were extracted using online available target prediction tools. Differentiated C2C12 cells were transfected with the miR-449a mimic and/or its inhibitor and the levels of the target mRNA and protein was evaluated by qRT-PCR and Western Blot analysis. This was validated by luciferase wild type and mutated constructs of the target 3’UTR. Inhibition of Notch signalling was assessed by evaluating the transcript levels of Notch target genes, Hes1 and Hey1 and the status of NICD (Notch Intracellular domain) by immunofluoresence microscopy. Effect of miR-449a on insulin signalling was evaluated by monitoring insulin induced PI3K and AKT phosphorylation and glucose uptake.ResultsOur data demonstrate that in C2C12 skeletal muscle cells, miR-449a binds to the 3’UTR of Jag1, an important Notch ligand, and down-regulates, both its transcript and protein levels. This was, however, prevented in the presence of the miR-449a inhibitor that suggests the specificity of the miRNA effect. This was validated in human primary skeletal muscle cells where miR-449a decreased Jag1 protein levels and this was prevented in the presence of the miR-449a inhibitor. This miR-449a-Jag1 interaction subsequently affects the Notch signalling pathway as was evident by the fact that miR-449a decreased the levels of NICD and consequently, the levels of Notch target genes, Hes1 and Hey1 were significantly inhibited. miR-449a and Notch pathway inhibition using DAPT, significantly increased insulin stimulated PI3K and AKT phosphorylation and these were prevented in the presence of the miR-449a inhibitor.ConclusionOur results indicate towards a critical role for miR-449a and its target, Jag1 in regulating Notch signalling and insulin signalling in the skeletal muscle and imply that targeting this axis might hold therapeutic potential for impaired skeletal muscle metabolism during diabetes.

Highlights

MiR-449a, an intronic miRNA, is highly down-regulated in the skeletal muscle during diabetes
Jag1 harbors a specific binding site for miR-449a in its 3’untranslated region (UTR) (Fig. 1c) and this interaction is conserved across species (Fig. 1d) which justifies that it might be targeted by miR-449a
In cells transfected with the miR-449a inhibitor alone, cellular levels of Jag1 significantly increased at the doses 10 and 25 nM (Fig. 2d). All these suggest that miR-449a possibly targets Jag1 and decreases its levels. This was validated in primary human skeletal muscle cells and as shown in Fig. 2e, miR-449a significantly decreased Jag1 protein levels and this was prevented in the presence of the miR-449a inhibitor

Summary

Introduction

MiR-449a, an intronic miRNA, is highly down-regulated in the skeletal muscle during diabetes. MicroRNAs (miRNAs) are small (~ 22 nucleotides) endogenous RNA sequences that regulate gene expression by primarily binding to the corresponding target mRNA 3′ UTR sequence and promoting mRNA degradation and/or translational repression. Their expression is controlled mainly at the transcriptional level [1,2,3] wherein epigenetic modifications play a crucial role in determining their cellular levels [4, 5]. Silencing of miR-199a* in several cancer cell lines occurs due to hypermethylation of its gene [7] This miRNA inhibits the proto-oncogene, MET and its downstream effector, ERK2 and this interaction affects cell proliferation, motility and invasive capabilities of tumor cells

Methods

Results

Discussion

Conclusion