The Role of Microrna Let-7g in Cytosolic Ca2+ Regulation and Cox-2 Gene Expression

Atherosclerosis, a disease of the large arteries, is the primary cause of cardiovascular diseases. Previous results have indicated that oxLDL can trigger vascular smooth cell proliferation via miRNA let-7g expression. Clinical evidences suggested close correlation between miRNA let-7g expression and the development of atherosclerosis. Our preliminary results indicated that stimulation of high dose let-7g increased cellular calcium concentration which involved in the regulation of inflammatory COX-2 gene. However, the molecular mechanism is still unclear. In this proposal, three specific aims will be tested : (1) to determine the major calcium channel regulated by miRNAlet-7g (2) to explore the molecular mechanism of miRNA let-7g-mediated COX-2 gene expression (3).to understand whether epigenetic modifications by miRNA let-7g influence transcription factors and COX-2 promoter. We plan to use small interfering RNA approaches and pharmacological inhibitors to knock down store-operated calcium channel, and TRPC ion channels superfamily (TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7). We will correlate the effects of let-7g-induced Ca2+ mobilization and COX-2 gene expression. We also will use different COX-2 promoter mutants to further study the mechanism of how let-7g -mediated cellular signaling pathways. Epigenetics modification in let-7-mediated transcription factors will also be addressed. The proposal is innovative because 1) our study will allow us to understand the molecular mechanisms of how let-7g regulates physiological calcium changes and COX-2 gene activation, which may facilitate us to find potential targets for drug development in anti-inflammatory diseases; 2). Using a combination of molecular biological tools (COX-2 mutants, RNAi) and physiological approaches (Ca2+ imagine), our proposal will lead to a better understanding of clinical application in atherosclerosis and stroke.