MicroRNA-99a mimics inhibit M1 macrophage phenotype and adipose tissue inflammation by targeting TNFα.

Cellular & molecular immunology (2018-06-01)
Anant Jaiswal, Sukka Santosh Reddy, Mohita Maurya, Preeti Maurya, Manoj Kumar Barthwal

In human adipose tissue and obesity, miR-99a expression is negatively correlated with inflammation. Therefore, the present study investigated the role of miR-99a in macrophage phenotype activation and adipose tissue inflammation. M2 BMDMs showed a significant increase in miR-99a expression when compared to the M0 and M1 phenotypes. Phenotype-switching experiments established an association between upregulated miR-99a expression and the M2 phenotype. Overexpression of miR-99a prevented M1 phenotype activation and attenuated bactericidal activity. Likewise, knockdown of miR-99a abolished M2 phenotype activation. By means of in silico target prediction tools and a luciferase reporter assay, TNFα was identified as a direct target of miR-99a. Knockdown of TNFα recapitulated the effect of miR-99a overexpression in M1 BMDMs. In a db/db mice model, miR-99a expression was reduced in eWAT and F4/80+ ATMs. Systemic overexpression of miR-99a in db/db mice attenuated adipocyte hypertrophy with increased CD301 and reduced CD86 immunostaining. Flow cytometry analysis also showed an increased M2 and a reduced M1 macrophage population. Mimics of miR-99a also improved the diabetic dyslipidemia and insulin signaling in eWAT and liver, with an attenuated expression of gluconeogenesis and cholesterol metabolism genes in the liver. Furthermore, adoptive transfer of miR-99a-overexpressing macrophages in the db/db mice recapitulated in vivo miR-99a mimic effects with increased M2 and reduced M1 macrophage populations and improved systemic glucose, insulin sensitivity, and insulin signaling in the eWAT and liver. The present study demonstrates that miR-99a mimics can regulate macrophage M1 phenotype activation by targeting TNFα. miR-99a therapeutics in diabetic mice reduces the adipose tissue inflammation and improves insulin sensitivity.

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DAPI, for nucleic acid staining
MISSION® pLKO.1-puro Empty Vector Control Plasmid DNA, Contains no shRNA insert