In Hdl Receptor Sr - Bi Target Gene 3 'utr Of Mirnas And The Discovery Of Small Molecule Compounds And Mechanism Research

Despite significant improvements in the cardiovascular health in recent decades, cardiovascular disease (CVD) continues to be the leading cause of morbidity and mortality worldwide. It imposes a significant burden in terms of costs, premature death, long-term disability, restricted social functioning and reduction in patient’s quality of life. Effective interventions in cardiovascular disease treatment and prevention have long-term clinical and economic consequences. Since atherosclerosis is the central biological process underlying most cardiovascular diseases, new and better anti-atherosclerotic strategies are required. Plasma concentrations of high density lipoprotein (HDL) have been known to inversely correlate with risk for atherosclerosis. Bulk transport of HDL cholesterol from the peripheral tissues to the liver is a major pathway, termed reverse cholesterol transport, responsible for maintaining whole body cholesterol homeostasis. A key mechanism of receptor-mediated direct delivery of HDL cholesteryl esters to the liver and steroidogenic tissues is selective cholesterol uptake. Scavenger receptor class B type I (SR-BI), HDL receptor, mediates the selective uptake of plasma HDL cholesterol by the liver and steroidogenic tissues. Increasing the expression of hepatic SR-BI promotes reverse cholesterol transport and reduces atherosclerosis. Therefore, SR-BI has considerable antiatherogenic capacity. SR-BI may become an attractive target for prevention of or therapeutic intervention in a variety of human diseases.In the first part of this study, the potential involvement of microRNAs in posttranscriptional regulation of hepatic SR-BI and selective HDL-C uptake was investigated. The level of SR-BI expression was repressed by miR-185, miR-96, and miR-223, while the uptake of DiI-HDL was decreased by31.9%(P<0.001),23.9%(P <0.05), and15.4%(P<0.05), respectively, in HepG2cells. The inhibition of these microRNAs by their anti-microRNAs had opposite effects in these hepatic cells. The critical effect of miR-185was further validated by the loss of regulation in constructs with mutated miR-96, miR-185and miR-223target sites. In addition, these microRNAs directly targeted the3’untranslated region (UTR) of SR-BI with a coordinated effect. Interestingly, the decrease of miR-96and miR-185coincided with the increase of SR-BI in the livers of ApoE KO mice on a high-fat diet. These data suggest that miR-185, miR-96, and miR-223may repress selective HDL-C uptake through the inhibition of SR-BI in human hepatic cells, implying a novel mode of regulation of hepatic SR-BI and an important role of microRNAs in modulating cholesterol metabolism.In the second part of this study, we discovered that several compounds increased SR-BI mRNA stability and confirmed they are regulators of SR-BI expression and function. To facilitate screening of compounds that increased SR-BI mRNA stability in a3’-untranslated region (3’UTR) relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an LUC-3’UTR fusion construct, in which firefly luciferase reporter mRNAs containing the SR-BI3’UTR (LUC-3’UTR). The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. The chemical library containing over25000compounds was screened and analyzed. Compound hits identified by HTS were further evaluated. The effects on SR-BI expression and function were measured in HepG2cells. Six compounds were confirmed to significantly increase SR-BI expression and DiI-HDL uptake. The positive rate of HTS was0.23%o. Treatment with the compound5172B-41or compound5238B-63significantly increased SR-BI mRNA stability, since they lengthened SR-BI mRNA half-life and increased SR-BI mRNA and protein levels. Further studies showed that treatment with both compound5172B-41and compound5238B-63significantly increased expression of SR-BI expression and DiI-HDL uptake in human HepG2cells in a concentration and time dependent manner. To obtain better candidate compounds for future studies, structure-activity relationship analysis was performed. Our hit compounds could be ideal starting points for the development of new drug candidates or lead compounds to treat cardiovascular diseases.