CARMN, a novel smooth muscle-specific lncRNA, is a critical regulator of smooth muscle phenotype

In many vascular diseases such as post-angioplasty restenosis and atherosclerosis, vascular smooth muscle cells (VSMCs) undergo phenotypic switching from a contractile to a proliferative phenotype that results in increased proliferation and migration and decreased expression of SM-specific contractile proteins. Identification of the key regulators involved in SM phenotypic switching will lead us towards better understanding the pathology of these diseases and ultimately designing therapeutic agents for their treatment and prevention. Human genome encodes thousands of long noncoding RNAs (lncRNAs) and emerging evidence suggests that many of them act as critical regulators of gene expression programs in diverse biological processes. However, their expression and functions in VSMCs and vascular diseases are largely unknown. In an effort to identify lncRNAs with a role in regulating VSMC phenotype, we used publicly available RNA-seq data that are generated from different cell and tissue types to screen VSMC-enriched lncRNAs. This unbiased whole transcriptome analysis revealed that the lncRNA, CARMN (Cardiac mesoderm enhancer-associated noncoding RNA), is specifically expressed in VSMCs. Our preliminary data showed that the enrichment of CARMN expression in SMCs is dependent on serum response factor (SRF)/myocardin (MYOCD) complex, a central regulator of SM-specific genes. We further found CARMN promoter contained two evolutionarily conserved SRF binding sites and can be trans-activated by MYOCD. In addition, I demonstrated that CARMN is down-regulated during VSMC phenotypic switching both in vitro and in vivo. More importantly, over-expression of CARMN activates expression of VSMC contractile genes, while suppresses the proliferation of VSMCs. Therefore, experiments described in this proposal will test the novel hypothesis that the SRF/MYOCD-mediated SMC-enriched lncRNA CARMN plays a critical role in regulating VSMC phenotypic switching and preventing neointima hyperplasia following arterial injury. This hypothesis will be tested by addressing the following two specific aims: 1) To test the hypothesis that SRF/MYOCD directly regulates CARMN expression by ChIP assay, mutagenesis and luciferase reporter assay. 2) To test the hypothesis that overexpression of CARMN will attenuate neointima hyperplasia in vivo by infusing adenovirus expressing CARMN into rat balloon injury carotid artery and assessing the lesion formation. (AHA Program: Postdoctoral Fellowship)