Plasma high LDL and low HDL cholesterol levels are risk factors for cardiovascular diseases. MicroRNAs (miRs), small non-coding endogenous RNAs, target multiple pathways and regulate physiologic functions. Because miRs modulate diverse biological pathways, we hypothesized that miRs exist in nature that might simultaneously lower LDL and increase HDL. To discover them, we screened a human miRs library and identified miR-1200 that decreased apoB (LDL scaffold protein) and increased apoAI (major HDL protein) secretion from human hepatoma cells. We propose to find out how miR-1200 decreases apoB secretion, increases apoAI secretion, and enhances hepatic fatty acid oxidation (FAO) to modulate plasma lipoproteins and reduce atherosclerosis without causing hepatosteatosis in mice.
Aim 1 : Physiological mechanisms modulating plasma lipoproteins and atherosclerosis in mice. (A) We will interrogate the effects of miR-1200 on plasma lipoproteins and atherosclerosis in Apoe?/? and Ldlr?/? mice. Further comprehensive changes in weight gain, VO2, VCO2, activity and feeding parameters as well as in plasma lipids, lipoproteins, glucose, cytokines, chemokines and transaminases will be determined. Accretions of miR-1200 in different tissues and its consequences on target and control genes, hepatic lipids, lipid synthesis and fatty acid oxidation (FAO) will be studied. Additionally, we will evaluate whether miR-1200 is able to prevent diet induced obesity in wild type mice fed a high fat diet. (B) We will evaluate the hypothesis that miR-1200 regulates hepatic apoB and apoAI secretion to modulate plasma LDL and HDL levels. (C) We will test the hypothesis that miR-1200 modulates plasma LDL and HDL levels, and hepatic FAO by regulating APOB, BCL11B and NCOR1 gene expression. (D) We will ask if the elevated HDL is competent in cholesterol efflux and reverse cholesterol transport and is anti-inflammatory and anti- oxidant. We foresee that increasing hepatic miR-1200 levels will (1) reduce plasma LDL, (2) augment HDL, (3) increase reverse cholesterol transport, and (4) enhance FAO. Via these mechanisms, miR-1200 will reduce atherosclerosis and diet-induced obesity.
Aim 2 : Molecular mechanisms regulating lipid metabolism by miR-1200: (A) Mechanisms decreasing apoB secretion: We will (1) measure mRNA and protein levels in human primary hepatocytes transfected with miR-1200 or Control miR, (2) determine if miR-1200 interacts with seed sequence to enhance posttranscriptional degradation of apoB mRNA. (B) Mechanisms increasing apoAI secretion: We hypothesize that miR-1200 reduces BCL11B expression, a repressor, to increase ApoaI transcription. Experiments will be conducted to (1) establish that BCL11B is regulated by miR-1200, (2) demonstrate if BCL11B represses apoAI expression, (3) identify the binding site(s) for BCL11B in the ApoaI promoter, and (4) determine whether BCL11B binds less to the ApoaI promoter in miR-1200?expressing cells. These studies will identify a novel mechanism of regulating apoAI expression. (C) Mechanisms increasing FAO: We hypothesize that miR-1200 enhances FAO by repressing NCOR1, a repressor. We will establish that miR-1200 interacts with the 3?-UTR of NCOR1 mRNA and enhances its degradation to increase FAO. Further, we will show that miR-1200 modulates the binding of NCOR1 to MCAD and CPT1 promoter. These studies will provide proof of concept that there are miRs that differentially regulate plasma lipoproteins. Further, they will explain molecular mechanisms involved in the regulation of plasma lipoproteins and hepatic FAO. These studies may show that miR-1200 can potentially lower or prevent diet- induced obesity and reduce atherosclerosis without causing steatosis and increasing plasma transaminases. These studies may point to the possibility that miR-1200 could be a potential drug to treat obesity and atherosclerosis.
Cardiovascular diseases are the number-one killers of Americans, and are the leading cause of hospitalization in the VA health care system. Since high plasma lipids, especially cholesterol, are a major risk factor for these diseases, new therapeutic agents are needed to lower their levels. The proposed studies may uncover a novel microRNA that regulates plasma concentrations of these lipids. Such studies may be useful in developing new drugs in the future to modulate plasma lipids.
