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

新型平滑肌特异性 lncRNA CARMN 是平滑肌表型的关键调节因子

• 批准号: 10543860
• 负责人: Jiliang Zhou
• 金额: $ 50.05万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-20 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543860
• 关键词: Area; Atherosclerosis; Attenuated; Binding; Bioinformatics; Biological Assay; Biological Process; CRISPR/Cas technology; Cardiac; Cardiovascular Diseases; Cardiovascular system; Carotid Arteries; Cells; ChIP-seq; Complex; Data; Data Set; Databases; Development; Disease; Elements; Event; Feedback; Foundations; Gene Expression; Genes; Genetic Transcription; Genomic DNA; Genotype-Tissue Expression Project; Goals; Human; Hyperplasia; Immunofluorescence Immunologic; In Situ Hybridization; In Vitro; Injury; Knock-in; Knock-in Mouse; Knock-out; Knockout Mice; Lesion; Link; Luciferases; Maps; Mediating; Modeling; Mus; Mutagenesis; Mutate; Pathogenesis; Phenotype; Physical assessment; Play; Quantitative Reverse Transcriptase PCR; RNA; Rattus; Reporter; Role; Serum Response Factor; Smooth Muscle; Smooth Muscle Myocytes; Testing; Therapeutic; Therapeutic Agents; Tissues; Transactivation; Transcript; Transcription Coactivator; Untranslated RNA; Vascular Diseases; Vascular Smooth Muscle; Western Blotting; cell type; chromatin immunoprecipitation; cofactor; design; experimental study; femoral artery; genome editing; genomic locus; in vivo; injured; insight; mortality; mouse development; myocardin; novel; overexpression; prevent; restenosis; transcriptome; transcriptome sequencing

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.

项目摘要 血管平滑肌细胞（VSMC）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，事件的关键因素 还远未完全确定。新出现的证据表明，长链非编码RNA（lncRNA）是至关重要的 用于基因表达，但VSMC特异性lncRNA仍然定义不清。为了鉴定lncRNAs在 为了调节VSMC表型，我们利用了公共可用的RNA-seq和ChIP-seq数据集， 以鉴定富含VSMC的lncRNA。这种无偏分析显示， CARMN在VSMC中特异性表达。相关性分析显示，SM特异性表达的 CARMN不仅与一组众所周知的SM收缩标志物相关，而且与血清反应相关。 因子（SRF）及其心脏/SM特异性辅因子心肌素（MYOCD）。SRF/MYOCD是一种转录复合物 通过与CArG盒结合，在调节SM特异性收缩基因表达中起关键作用 在这些基因中。生物信息学分析在CARMN基因中发现了2个进化上保守的CArG盒 基因座和我们令人兴奋的初步数据进一步表明，CARMN表达是SRF/MYOCD- 依赖。此外，我们发现CARMN在体内VSMC表型转换过程中下调， 和体外。更重要的是，CARMN的缺失抑制了细胞增殖，而CARMN的过表达促进了细胞增殖。 VSMCs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作为 通过结合MYOCD转录激活因子，但也协同增强MYOCD介导的 包括CARMN本身在内的SM特异性基因的反式激活。因此，我们假设SRF/MYOCD 驱动SM特异性lncRNA CARMN表达，CARMN在维持心肌收缩中起关键作用， 通过与MYOCD结合，通过正反馈机制调节VSMCs的状态。提出了三个目标来检验 这个新的假设。在目标1中，我们将确定CARMN特异性的调节机制， 通过使用我们的新型CARMN基因敲入报告小鼠、ChIP测定、体外诱变和 体内CArG盒的CRISPR-Cas9基因组编辑。在目标2中，我们将定义CARMN在以下方面的功能作用： 使用我们的新的可诱导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VSMCs的物理和功能来探索CARMN在VSMCs中的作用机制。 CARMN与MYOCD的结合。这些研究的完成将提供新的见解的机制 控制VSMC表型可塑性，并鉴定新的SM特异性lncRNA CARMN，用于治疗许多 增殖性血管疾病。

项目成果

YY1 directly interacts with myocardin to repress the triad myocardin/SRF/CArG box-mediated smooth muscle gene transcription during smooth muscle phenotypic modulation.

• DOI: 10.1038/s41598-020-78544-3
• 发表时间: 2020-12-11
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Zheng JP;He X;Liu F;Yin S;Wu S;Yang M;Zhao J;Dai X;Jiang H;Yu L;Yin Q;Ju D;Li C;Lipovich L;Xie Y;Zhang K;Li HJ;Zhou J;Li L
• 通讯作者: Li L