Hypercholesterolemia is a strong predictor of cardiovascular diseases that result in the largest number of mortality and morbidity worldwide. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase gene (Hmgcr) coding for the rate-limiting enzyme in the cholesterol biosynthesis pathway is an important candidate gene for essential hypertension (EH) and a crucial regulator of plasma cholesterol levels. However, the mechanisms of regulation of Hmgcr expression under basal and pathological conditions remain unclear. In the present study, we probed the Hmgcr transcript and protein levels in liver tissues of a genetic rodent model of EH viz. Spontaneously Hypertensive Rats (SHR) as well as its normotensive control WKY rats and observed diminished Hmgcr expression in SHR as compared to WKY in these tissues. Consistently, a number of other rat models of hypertension display diminished cholesterol levels as compared to their corresponding control strains. Sequencing of the Hmgcr promoter in SHR/WKY reveals three variations: A-405G, C-62T and a 11 bp insertion (-398_-388insTGCGGTCCTCC) in SHR. Moreover, SHR-Hmgcr promoter displays higher activity than WKY-Hmgcr promoter in various cell lines. Transient transfections of Hmgcr-promoter mutants and in silico analysis suggest altered binding of Runx3 and Srebf1 across A-405G. Indeed, chromatin immunoprecipitation assays confirm differential binding of Runx3/Srebf1 to Hmgcr promoter leading to diminished expression of Hmgcr in SHR as compared to WKY under basal/cholesterol-modulated conditions. Here, we also show that Hmgcr is markedly inhibited, while miR-27a is highly induced in various human tissues as well as several rodent models of metabolic disorders (viz. genetically hypertensive blood pressure high mice, spontaneously hypertensive rats and high fat and high fructose diet-fed rats). Our in vitro data shows that miR-27a specifically interacts with the 3-untranslated region of Hmgcr in murine and human hepatocytes. Actinomycin D chase assays and pulse-chase experiments demonstrate that miR-27a regulates Hmgcr by translational attenuation followed by mRNA degradation. Moreover, systematic in silico and functional analyses reveal that miR-27a expression is modulated by intracellular cholesterol level via Early Growth Response 1 transcription factor. Augmentation of miR-27a levels upon tail-vein injection of miR-27a mimic in Apoe-/- mice significantly lowers plasma LDL/total cholesterol, atherosclerotic plaque burden and improves cardiac function. Pathway and gene expression analyses reveal that miR-27a also targets other genes (apart from Hmgcr) involved in cholesterol homeostasis. Taken together, this study provides mechanistic insights for altered Hmgcr expression in rat models of EH, thereby unravelling the links of this gene to hypertension and highlights miR-27a as an attractive therapeutic candidate for clinical management of hypercholesterolemia and atherosclerosis.



Publications:

1. Sonawane P. J.*, Gupta V.*, Sasi B. K., Kalyani A., Natarajan B., Khan A. A., Sahu B. S., Mahapatra N. R. Transcriptional Regulation of the Novel Monoamine Oxidase Renalase: Crucial Roles of Transcription Factors Sp1, STAT3 and ZBP89. Biochemistry. 2014; 53 (44):6878-92 (* equal contribution).



2. Gupta V., Khan A. A., Sasi B. K., Mahapatra N. R. Molecular Mechanism of Monoamine Oxidase A Gene Regulation under Inflammation and Ischemia-like conditions: Key Roles for the Transcription Factors GATA2, Sp1 and TBP. J Neurochem. 2015; 134(1):21-38.



3. Kalyani A., Sonawane P. J., Khan A. A., Subramanian L., Ehret G. B., Mullasari A. S., Mahapatra N. R. Post-Transcriptional Regulation of Renalase Gene by miR-29 and miR-146 MicroRNAs: Implications for Cardiometabolic Disorders. J Mol Biol. 2015; 427(16):2629-46.



4. Gupta V., Kapopara P. R., Khan A. A., Arige V., Subramanian L., Sonawane P. J., Sasi B. K., Mahapatra N. R. Functional promoter polymorphisms direct the expression of cystathionine gamma-lyase gene in mouse models of essential hypertension. J Mol Cell Cardiol. 2017; 102:61- 72 (cover page article).



5. Subramanian L., Khan A. A., Allu P. K. R, Kiranmayi M., Sahu B. S., Sharma S, Khullar M., Mullasari A. S., Mahapatra N. R. A haplotype variant of the human chromogranin A gene (CHGA) promoter increases CHGA expression and the risk for cardiometabolic disorders. J Biol Chem. 2017; 292(34):13970-13985.



6. Arige V., Agarwal A., Khan A. A., Kalyani A., Natarajan B., Gupta V., Reddy S. S., Barthwal

M. K., Mahapatra N.R. Regulation of Monoamine Oxidase B Gene Expression: Key Roles for Transcription Factors Sp1, Egr1 and CREB, and microRNAs miR-300 and miR-1224. J Mol Biol. 2019 ;431(6):1127-1147.



7. Khan, A. A., Agarwal, H., Reddy, S. S., Arige, V., Natarajan B., Gupta, V., Kalyani, A., Barthwal M. K., Mahapatra N. R. MicroRNA-27a is a key modulator of cholesterol biosynthesis. Mol. Cell. Biol, 2020; 40 (9): e00470-19.