Identification, regulation and function of the smooth muscle-specific long non-coding RNA

Phenotypic switching of vascular smooth muscle cells (VSMCs) from a contractile to a proliferative phenotype, plays a causal role in many human occlusive vascular diseases. However, the key factors critical for SM phenotypic switching are far from completely identified. Emerging evidence suggests that long non-coding RNAs (lncRNAs) are critical for gene expression but VSMC-specific lncRNAs remain ill-defined. In an effort to identify lncRNAs with a role in regulating VSMC phenotype, we utilized publicly available RNA-seq and ChIP-seq data sets that are generated from different cells/tissues to identify VSMC-enriched lncRNAs. This unbiased analysis revealed that the lncRNA CARMN is specifically expressed in VSMCs. Correlation analysis revealed that SM-specific expression of CARMN is not only correlated with a set of well-known SM-contractile markers, but also with serum response factor (SRF) and its cardiac/SM-specific cofactor myocardin (MYOCD). SRF/MYOCD is a transcriptional complex that plays a critical role in regulating SM-specific contractile gene expression, through binding to the CArG boxes within these genes. Bioinformatic analysis identified 2 evolutionarily conserved CArG boxes within CARMN gene locus and our exciting preliminary data further demonstrated that CARMN expression is SRF/MYOCD-dependent. Furthermore, we found that CARMN is down-regulated during VSMC phenotypic switching in vivo and in vitro. More importantly, depletion of CARMN inhibits while overexpression of CARMN promotes the contractile phenotype of VSMCs. Experiments are proposed to test the novel hypothesis that SRF/MYOCD drives CARMN expression in VSMCs (Aim 1) & that CARMN plays a critical role in maintaining the contractile state of VSMCs (Aim 2). These hypothesis will be tested by using our novel CARMN knock-in reporter mice, ChIP assay, mutagenesis in vitro and in vivo; and by using the novel inducible SM-specific CARMN KO mice (loss-of-function) and infusing adenovirus expressing CARMN into balloon injured rat carotid artery (gain-of-function) to assess the lesion formation following arterial injury. (AHA Program: Transformational Project Award)