Edited by Laszlo NagyKeywords: MicroRNA Macrophage Lipoprotein Toll-like receptor 4 Atherosclerosis a b s t r a c t Atherosclerosis is an inflammatory process due to oxidized low-density lipoprotein (oxLDL) accumulation in macrophages. We investigated the involvement of microRNAs in oxLDL accumulation and inflammatory response in macrophages. The expression of miR-146a decreases under oxLDL stimulation. MiR-146a significantly reduces intracellular LDL cholesterol content and secretion of interleukin 6, interleukin 8, chemokine (C-C motif) ligand 2 and matrix metallopeptidase 9. Toll-like receptor 4 (TLR4) is a relevant target of miR-146a, and miR-146a inhibits the activation of TLR4-dependent intracellular signaling pathways involved in cytoskeleton rearrangement, lipid uptake, and inflammatory cytokine secretion. These results indicate that miR-146a contributes to the regulation of both oxLDL accumulation and inflammatory response by negatively regulating TLR4 and thereby inhibiting the activation of TLR4-dependent signaling pathways. Over-expression of miR146a may be useful in the prevention and treatment of atherosclerosis.

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BACKGROUND MicroRNAs are important regulators of gene expression, including those involving electrical remodeling in atrial fibrillation (AF). Recently, KCNN3, the gene that encodes the small conductance calcium-activated potassium channel 3 (SK3), was found to be strongly associated with AF. OBJECTIVES This study sought to evaluate the changes in atrial myocardial microRNAs in patients with permanent AF and to determine the role of microRNA on the regulation of cardiac SK3 expression. METHODS Atrial tissue obtained during cardiac surgery from patients (4 sinus rhythm and 4 permanent AF) was analyzed by microRNA arrays. Potential targets of microRNAs were predicted by software programs. The effects of specific microRNAs on target gene expression were evaluated in HL-1 cells from a continuously proliferating mouse hyperplastic atrial cardiomyocyte cell line. Interactions between microRNAs and targets were further evaluated by luciferase reporter assay and by Argonaute pull-down assay. RESULTS Twenty one microRNAs showed significant, greater than two-fold changes in AF. miR-499 was upregulated by 2.33 fold (P<0.01) in AF atria, whereas SK3 protein expression was down-regulated by 46% (P<0.05). Transfection of miR-499 mimic in HL-1 cells resulted in the downregulation of SK3 protein expression, while that of miR-499 inhibitor upregulated SK3 expression. Binding of miR-499 to the 3′UTR of KCNN3 was confirmed by luciferase reporter assay and by the enhanced presence of SK3 mRNA in Argonaute pulled-down microRNA-induced silencing complexes (mRISC) after transfection with miR-499. CONCLUSION Atrial miRNA-499 is significantly upregulated in AF, leading to SK3 downregulation and possibly contributing to the electrical remodeling in AF.

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