Identification of MicroRNAs Regulating Apolipoprotein Secretion.

Abstract

A major risk factor for cardiovascular disease (CVD) is plasma cholesterol that is carried in plasma by two lipoproteins LDL and HDL. Currently available therapies lower LDL and attempts are undergoing to enhance HDL levels for therapeutic purposes. With advances in microRNAs (miRs), small non-coding RNAs that affect multiple pathways, we hypothesize that there might be miRs that regulate plasma lipoproteins. The major proteins in LDL and HDL are apoB and apoA1. To identify miRs that regulate both LDL and HDL, I took two different approaches. In the first approach, I searched for miRs that could potentially modulate apoB and apoA1 expression. From TargetScan, I found that human apoB mRNA contained two predicted targeting sites for miR-548p. In both Huh-7 and HepG2 cells, overexpression of miR-548p significantly decreased intracellular and secreted medium apoB without affecting apoAI. Mechanistic studies revealed that miR-548p decreased apoB expression levels by destabilizing its mRNA. Luciferase reporter assay indicated that miR-548p interacts with apoB 3'-UTR. Site-directed mutagenesis revealed that both seed and supplementary sequences on Site II, not Site I, were required for the targeting of miR-548p on apoB 3'-UTR. Additionally, the cholesterol and fatty acid syntheses rates were decreased in miR-548p overexpressing Huh-7 cells. We also found that Hmgcr and ACSL4, two important enzymes in cholesterol and fatty acid synthesis process, were predicted targets of miR-548p. Indeed, the mRNA and protein levels of Hmgcr and ACSL4 were significantly lowered by miR-548p. The direct targeting of miR-548p on the 3'-UTR of Hmgcr and ACSL4 was studied by luciferase reporter assay. In summary, a novel miR-548p decreased apoB secretion from Huh-7 cells by targeting apoB 3'-UTR and promoting its posttranscriptional degradation. MiR-548p also reduces cholesterol and fatty acid synthesis by down-regulating Hmgcr and ACSL4 expression levels. In the second approach, I took an unbiased global approach to identify miRs that individually and simultaneously affect apoB and apoA1. I transfected human hepatoma Huh7 cells in duplicate with a library of 1237 miRs and measured apoB and apoA1 in the media. This resulted in the identification of 13 and 28 miRs that increased and decreased apoB secretion. In addition, I identified 11 and 23 miRs that increased and decreased apoA1 secretion. Most interestingly, I identified 3 miRs that simultaneously increased apoB and reduced apoA1 levels. I further characterized miR-1200 and explained mechanisms how it regulates apoB and apoA1 secretion. Mechanistic studies showed that miR-1200 reduced apoB secretion by lowering the mRNA levels of apoB. In addition, post-transcriptional degradation of apoB was promoted in miR-1200 transfected cells. Bioinformatic analysis revealed apoB as a predicted target of miR-1200. Luciferase reporter assay and site-directed mutagenesis were used to confirm the direct miR-mRNA interaction. In contrast, miR-1200 increased apoAI protein and mRNA levels by increasing its de novo transcription. The mechanistic studies resulted in the identification of a novel transcription repressor of apoAI, BCL11B. Therefore, miR-1200 increases the transcription of apoAI by reducing the expression levels of BCL11B, and promotes the secretion of apoAI. I also observed that the overexpression of miR-1200 in Huh-7 cells decreased lipid synthesis, which might avoid the lipid accumulation in the liver. In summary, these studies identified a novel human microRNA-1200 that could potentially reduce apoB-containing lipoproteins and increase HDL production from the liver, as well as decreasing lipid synthesis in human hepatoma cell line. These three different roles of miR-1200 in lipid metabolism regulation suggest that it may have protective function against atherosclerosis. In short, I have identified two novel miRs-548p and 1200 as potent regulators of apolipoprotein and lipid metabolism in hepatoma cells. Both have the potential to be developed as therapeutic agents for the treatment of dyslipidemia and atherosclerosis while avoiding hepatic steatosis. As these are primate specific miRs, the physiological effect of overexpressing or inhibiting endogenous miR-548p or miR-1200 could be tested in humanized mice or non-human primate models.