Enhanced levels of microRNA-125b in vascular smooth muscle cells of diabetic db/db mice lead to increased inflammatory gene expression by targeting the histone methyltransferase Suv39h1.
OBJECTIVE
Diabetes remains a major risk factor for vascular complications that seem to persist even after achieving glycemic control, possibly due to "metabolic memory." Using cultured vascular smooth muscle cells (MVSMC) from type 2 diabetic db/db mice, we recently showed that decreased promoter occupancy of the chromatin histone H3 lysine-9 methyltransferase Suv39h1 and the associated repressive epigenetic mark histone H3 lysine-9 trimethylation (H3K9me3) play key roles in sustained inflammatory gene expression. Here we examined the role of microRNAs (miRs) in Suv39h1 regulation and function in MVSMC from diabetic mice.


METHODS
We used luciferase assays with Suv39h1 3'untranslated region (UTR) reporter constructs and Western blotting of endogenous protein to verify that miR-125b targets Suv39h1. We examined the effects of Suv39h1 targeting on inflammatory gene expression by quantitative real time polymerase chain reaction (RT-qPCR), and H3K9me3 levels at their promoters by chromatin immunoprecipitation assays.


RESULTS
We observed significant upregulation of miR-125b with parallel downregulation of Suv39h1 protein (predicted miR-125b target) in MVSMC cultured from diabetic db/db mice relative to control db/+. miR-125b mimics inhibited both Suv39h1 3'UTR luciferase reporter activity and endogenous Suv39h1 protein levels. Conversely, miR-125b inhibitors showed opposite effects. Furthermore, miR-125b mimics increased expression of inflammatory genes, monocyte chemoattractant protein-1, and interleukin-6, and reduced H3K9me3 at their promoters in nondiabetic cells. Interestingly, miR-125b mimics increased monocyte binding to db/+ MVSMC toward that in db/db MVSMC, further imitating the proinflammatory diabetic phenotype. In addition, we found that the increase in miR-125b in db/db VSMC is caused by increased transcription of miR-125b-2.


CONCLUSIONS
These results demonstrate a novel upstream role for miR-125b in the epigenetic regulation of inflammatory genes in MVSMC of db/db mice through downregulation of Suv39h1.
Villeneuve
Louisa M
LM
Department of Diabetes, Beckman Research Institute of City of Hope, Duarte, California, USA.
Kato
Mitsuo
M
Reddy
Marpadga A
MA
Wang
Mei
M
Lanting
Linda
L
Natarajan
Rama
R
D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics; D015536:Down-Regulation; D005786:Gene Expression Regulation; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics*; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000517:prevention & control; D008780:Methyltransferases / Q000235:genetics*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000503:physiopathology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D015854:Up-Regulation
D020413:3' Untranslated Regions; D035683:MicroRNAs; D012097:Repressor Proteins; C407843:SUV39H1 protein, human; D000076983:Histone Methyltransferases; D008780:Methyltransferases; D011495:Histone-Lysine N-Methyltransferase