ABSTRACT
3-Hydroxy-3-methylglutaryl-coenzyme A reductase gene (Hmgcr) that codes for the rate-limiting enzyme in the cholesterol biosynthesis pathway is a key modulator of dyslipidemia and consequent cardiovascular diseases. However, mechanism of regulation of Hmgcr, especially at the post-transcriptional level, is poorly understood. In silico predictions coupled with systematic functional analysis revealed specific interactions of miR-27a with mouse Hmgcr 3’-UTR in mouse and human hepatocytes. In corroboration, miR-27a expression negatively correlated with Hmgcr transcript level in various cultured cell lines as well as rodent and human tissues. Hmgcr protein level also displayed inverse correlation with miR-27a level in liver tissues of several rodent models of metabolic disorders (viz. genetically hypertensive blood pressure high vs. genetically hypotensive blood pressure low mice, spontaneously hypertensive rats vs. Wistar Kyoto rats, and rats fed with high fat and fructose diet vs. rats fed with a normal chow diet). Consistently, ribonucleoprotein immunoprecipitation assays using antibodies against Ago2 and human hepatocyte HuH-7 cells over-expressing miR-27a revealed enrichment of HMGCR in the Ago2-immunoprecipitated fraction. Of note, cholesterol depletion in mouse hepatocyte AML-12 cells down-regulated endogenous miR-27a and augmented Hmgcr protein level; exogenous cholesterol treatment enhanced miR-27a with concomitant reduction in the Hmgcr protein level. Computational analysis of the proximal 1 kb promoter region of mmu-miR-27a predicted multiple Egr1 binding sites; corroboratively, over-expression/down-regulation of Egr1 augmented/diminished the miR-27a promoter activity in AML12 cells and chromatin immunoprecipitation (ChIP) assays confirmed in vivo interaction of Egr1 with the mmu-miR-27a promoter regions. Hypoxic stress augmented miR-27a and Egr1 expression in AML-12 cells; ChIP assays revealed enhanced binding of Egr1 with miR-27a promoter during hypoxia. Taken together, this study provides evidence for post-transcriptional regulation of Hmgcr by miR-27a under basal/pathophysiological conditions and has implications for understanding the mechanisms of cholesterol homeostasis.