oa Srebp-2 Intronic Microrna 33a Post-translationally Controls Ldl Uptake

Background and Objectives Impaired cholesterol and fat metabolism contributes to obesity, type 2 diabetes and atherogenic cardiovascular disease; major chronic conditions that are increasingly prevalent in Qatar. There is thus an urgent need for new treatment modalities to combat the rise in these diseases. Understanding how cholesterol/lipid homeostasis is achieved and maintained is among the very first critical steps towards developing new strategies for better treatments. Despite the intricate underlying mechanisms, many regulatory factors have been found to be involved in the metabolic regulation of lipids. Recent studies found that microRNAs (miR), short 22-nucleotide RNA molecules that control gene expression by silencing target mRNAs at the translational level, hold promise as therapeutic targets since they contribute to the etiology of many pathological conditions. We found that microRNA 33a (miR-33a) embedded within the intronic sequence of SREBP-2 gene, the master regulator of cholesterol metabolism, acts as a key modulator of intracellular cholesterol trafficking pathways. Here, we report our new findings on the 5p and 3p strands of miR-33a (miR-33a-5p and miR-33a-3p) in regulating cholesterol homeostasis. Methods Using computational algorithms we identified potential target genes of both strands of miR-33a that are involved in cholesterol metabolism. We then used luciferase reporter assays, wherein the 3'-UTR of the target gene was fused to a luciferase reporter gene, to validate the predicted targets of miR-33a. Positive associations between the microRNAs and their targets were further confirmed by mutating the binding sites on the target gene 3'-UTR and performing luciferase assays with the mutant constructs. Overexpression and knockdown of the microRNAs in human liver cell lines were carried out with mimics and inhibitors, respectively, and their effect on target gene expression was studied by RT-qPCR and western blotting. Cholesterol uptake and efflux assays in hepatocytes were exploited to better assess the functional roles of miR-33a. Results Intriguingly, we found that in addition to our pervious discovery of miR-33a-5p reducing cholesterol efflux from hepatocytes and macrophages, miR-33a-3p promotes intracellular cholesterol uptake. While miR-33a-5p inhibits ABCA1, a cholesterol efflux pump instrumental in raising plasma HDL-cholesterol levels and reverse cholesterol transport, miR-33a-3p activates LDL-receptor (LDLR) by repressing both Idol and PCSK9, two major negative regulators of LDLR. Accordingly, in an SREBP-2 active condition antisense-mediated inhibition of miR-33a-3p, but not miR-33a-5p, led to decreased LDLR expression and LDL uptake in human hepatocytes. Conclusions Mutations in the LDLR gene are well known to be highly associated with atherosclerosis and familial hypercholesterolemia. Our data unravels a novel regulatory circuit by which LDLR, which is transcriptionally activated by SREBP-2, is further protected from degradation at the post-translational level by miR-33a-3p. This indicates that both the 5p and 3p strands of miR-33a are mutually exclusive in elevating intracellular cholesterol levels along with their SREBP-2 host gene. These findings provide impetus for further characterization and development of new therapeutic interventions in cardiovascular disease.