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

项目总结 血管平滑肌细胞(VSMCs)从收缩表型到增殖表型的表型转换， 在许多人类闭塞性血管疾病中起着因果作用。然而，对这一事件至关重要的关键因素 还远未完全确定。新的证据表明，长的非编码RNA(LncRNAs)是至关重要的 对于基因表达，但VSMC特异性的LncRNAs仍然没有明确的定义。为了找出与LncRNA有关的 调节VSMC表型，我们利用了公开可用的RNA-SEQ和CHIP-SEQ数据集 从不同的细胞/组织中鉴定VSMC丰富的lncRNAs。这种不偏不倚的分析表明，lncRNA CARMN在VSMC中特异表达。相关分析表明，SM特异性表达 CARMN不仅与一组众所周知的SM-收缩标志物相关，而且与血清反应有关 因子(SRF)及其心脏/SM特异性辅因子肌钙蛋白(MYOCD)。SRF/MYOCD是一个转录复合体 它通过与Carg盒结合，在调节SM特异性收缩基因的表达中起着关键作用 在这些基因中。生物信息学分析鉴定CARMN基因内2个进化保守的CARG盒 进一步证明了CARMN的表达是SRF/MYOCD- 依附的。此外，我们还发现CARMN在体内VSMC表型转换过程中下调 而且是在体外。更重要的是，CARMN的耗竭抑制而CARMN的过表达促进 血管平滑肌细胞的收缩表型。值得注意的是，我们令人兴奋的初步数据进一步表明，CARMN起着 一种与MYOCD结合并协同增强MYOCD介导的转录激活剂 对SM特异基因的反式激活，包括CARMN本身。因此，我们假设SRF/MYOCD 驱动SM特异性LncRNA CARMN的表达，CARMN在维持收缩中起关键作用 通过与MYOCD结合的正反馈机制来评估VSMC的状态。提出了三个测试目标 这个新奇的假说。在目标1中，我们将确定CARMN具体通过的监管机制 利用我们新的CARMN敲入报告小鼠在VSMCs中表达，芯片检测，体外诱变和 体内Carg box的CRISPR-Cas9基因组编辑。在目标2中，我们将定义CARMN在 使用我们新的可诱导的SM特异性CARMN KO小鼠和大鼠颈动脉球囊损伤模型。 在目标3中，我们将通过评估生理和功能来探讨CARMN在VSMC中的作用机制 CARMN与MYOCD的结合。这些研究的完成将为研究这些机制提供新的见解 VSMC表型可塑性的控制及治疗多发性硬化症的新型SM特异性lncRNA CARMN的鉴定 增殖性血管疾病。

PROJECT SUMMARY 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 the event 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. Remarkably, our exciting preliminary data further showed that CARMN acts as a transcriptional activator by binding to MYOCD but also synergistically enhancing MYOCD-mediated transactivation on SM-specific genes including CARMN itself. Therefore, we hypothesize that SRF/MYOCD drives SM-specific lncRNA CARMN expression and that CARMN plays a critical role in maintaining the contractile state of VSMCs through a positive feedback mechanism by binding to MYOCD. Three aims are proposed to test this novel hypothesis. In Aim 1, we will determine the regulatory mechanism by which CARMN specifically expresses in VSMCs by using our novel CARMN knock-in reporter mice, ChIP assay, mutagenesis in vitro and CRISPR-Cas9 genome editing of CArG box in vivo. In Aim 2, we will define the functional role of CARMN in VSMCs by using our novel inducible SM-specific CARMN KO mice and rat carotid artery balloon injury model. In Aim 3, we will explore the mechanism of CARMN's action in VSMCs by assessing the physical and functional binding of CARMN with MYOCD. Completion of these studies will provide novel insights into the mechanisms controlling VSMC phenotypic plasticity and identify the novel SM-specific lncRNA CARMN for treating many proliferative vascular diseases.