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）从收缩到增殖表型的表型转换， 在许多人类闭塞性血管疾病中起因果作用。但是，事件至关重要的关键因素 远非完全确定。新兴的证据表明，长的非编码RNA（LNCRNA）很关键 对于基因表达，但VSMC特异性的lncRNA仍然不明确。为了识别具有角色的lncrnas 调节VSMC表型，我们使用了生成的公开可用的RNA-Seq和芯片序列数据集 从不同的细胞/组织来识别富含VSMC的LNCRNA。这种公正的分析表明lncRNA CARMN在VSMC中特别表达。相关分析表明，SM特异性表达 CARMN不仅与一组知名的SM取消标记相关，而且还与血清反应相关 因子（SRF）及其心脏/SM特异性辅因子心肌（Myocd）。 SRF/MYOCD是转录复合物 通过与carg盒结合，这在调节SM特异性收缩基因表达中起着至关重要的作用 在这些基因中。生物信息学分析确定了CARMN基因中的2个进化保守的carg盒 基因座和我们令人兴奋的初步数据进一步表明CARMN的表达是SRF/MYOCD- 依赖。此外，我们发现在VSMC表型切换体内，CARMN被下调 并在体外。更重要的是，CARMN的耗竭抑制，而CARMN的过表达促进了 VSMC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充当 通过与MyOCD结合，但也协同增强MyOCD介导的转录激活剂 在包括CARMN本身在内的SM特异性基因上进行反式激活。因此，我们假设SRF/MyOCD 驱动SM特异性LNCRNA CARMN表达，Carmn在保持收缩方面起着关键作用 通过与MyOCD结合，通过积极反馈机制的VSMC状态。提出了三个目标来测试 这个新颖的假设。在AIM 1中，我们将确定CARMN专门的调节机制 通过使用我们的新型CARMN敲入记者小鼠，芯片测定，体外诱变和 CRISPR-CAS9体内Carg Box的基因组编辑。在AIM 2中，我们将定义CARMN的功能作用 VSMC使用我们的新型诱导型SM特异性Carmn KO小鼠和大鼠颈动脉球囊损伤模型。 在AIM 3中，我们将通过评估物理和功能来探讨Carmn在VSMC中的作用机制 Carmn用MyOCD结合。这些研究的完成将为机制提供新的见解 控制VSMC表型可塑性并识别新型SM特异性LNCRNA CARMN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