Abstract 9687: Network Analysis Identifies MicroRNA-22 as a Key Regulator of Vascular Calcification
MicroRNAs (miR) have been shown to regulate expression of individual signaling molecules involved in vascular calcification. We hypothesized that downregulation of select miRs would modulate a calcification program in human coronary artery smooth muscle cells (SMC) to influence mineralization. To identify candidate miRs, we used a systems biology approach and incorporated 246 calcification-related genes into a network that revealed 3,013 interactions. A hypergeometric analysis found 9 miRNAs that regulate the network with >90% probability. Cross-referencing results with a miR microarray identified miR22 as a key regulator of the calcification network. To confirm the in silico analysis, SMC were transfected with anti-miR22 to decrease miR22 expression, with pre-miR22 to force miR22 expression, or with scrambled controls. Transfection with anti-miR22 increased SMC Ca2+ levels by 126% (p<0.05), and calcification by Alizarin red staining, while transfection with pre-miR22 inhibited SMC calcification. To determine how pro-calcifying mediators decrease miR22 levels, we examined c-myc, which negatively regulates miR expression. Chromatin immunoprecipitation assay demonstrated that c-myc bound to the miR22 promoter; SMC transfection with c-myc siRNA abrograted this response and increased miR22 levels. Next, we examined expression of miR22 targets identified by the network. Transfection of SMC with anti-miR22 increased expression of cysteine-rich angiogenic protein 61 (CYR61) by 154% (p<0.01), Smad4 by 68% (p<0.04), and its downstream target Runx2 by 112% (p<0.01). Upregulation of these proteins was inhibited by forced expression of miR22. In an ex vivo rat aorta calcification assay, transfection of vessels with anti-miR22 increased vascular CYR61 expression and calcification, while transfection with pre-miR22 inhibited calcification. In human coronary arteries, in situ hybridization revealed increased levels of miR22 in vessels with limited calcification as compared to heavily calcified vessels with significant atherosclerotic disease. These findings demonstrate that miR22 modulates expression of key calcification-related intermediaries. Therapies that target this signaling pathway may limit vascular calcification.
- © 2013 by American Heart Association, Inc.