Enhancer-associated LncRNA-ITGA2 promotes vascular smooth muscle remodeling by mediating ITGA2 promoter-enhancer interactions

In this study, multiomics profiling, including CUT&Tag, promoter capture Hi-C (PCHi-C), and microarray, identified LncRNA-ITGA2 as a novel elncRNA that was highly expressed in PDGF-induced proliferative human VSMCs. Notably, LncRNA-ITGA2 was significantly increased in both plasma and coronary atherosclerotic tissues of patients with coronary artery disease (CAD) compared with control subjects. Loss- and gain-of-function studies revealed that LncRNA-ITGA2 potently increased PDGF-induced VSMC proliferation and migration. Moreover, LncRNA-ITGA2 overexpression enhanced neointimal hyperplasia in vivo in a mouse carotid artery injury model, exhibiting its partial functional conservation. RNA-sequencing and CRISPR-Cas9 gene-editing technology revealed integrin α2 (ITGA2) as a downstream target of LncRNA-ITGA2 in VSMCs. Mechanistically, promoter-enhancer interactions were detected by PCHi-C at the ITGA2 locus after PDGF-BB treatment. Chromatin immunoprecipitation sequencing (ChIP-Seq) and chromatin isolation by RNA purification (ChIRP)-qPCR showed that LncRNA-ITGA2 directly bound to the Enhancer-ITGA2 and increased H3K27 acetylation in both the Enhancer-ITGA2 and Promoter-ITGA2. In addition, we demonstrated, by ChIRP-MS and RNA immunoprecipitation, that LncRNA-ITGA2 interacted with the DNA binding-protein NONO (non-pou domain containing octamer-binding protein), which also bound to the ITGA2 promoter, as verified by the ChIP-qPCR assay. Ultimately, the knockout cell lines of NONO, LncRNA-ITGA2, and its promoter confirmed the above regulatory mechanism. To investigate the DNA chromatin loops in vascular smooth muscle remodeling, promoter capture Hi-C was performed in human aortic smooth muscle cells treated with PDGF-BB or without PDGF-BB.

本研究通过涵盖CUT&Tag、启动子捕获Hi-C（PCHi-C）与基因芯片的多组学谱分析，鉴定出长链非编码RNA-ITGA2（LncRNA-ITGA2）为一类新型增强子相关长链非编码RNA（elncRNA），其在血小板衍生生长因子（PDGF）诱导的增殖性人血管平滑肌细胞（VSMCs）中呈高表达状态。值得注意的是，与对照受试者相比，冠状动脉疾病（CAD）患者的血浆与冠状动脉粥样硬化组织中LncRNA-ITGA2的表达量均显著升高。功能缺失与功能获得性实验表明，LncRNA-ITGA2可显著促进PDGF诱导的VSMC增殖与迁移。此外，在小鼠颈动脉损伤模型中，LncRNA-ITGA2过表达可增强内膜增生，体现出其部分功能保守性。通过RNA测序与CRISPR-Cas9基因编辑技术，研究人员证实整合素α2（ITGA2）是VSMCs中LncRNA-ITGA2的下游靶基因。机制层面，经PDGF-BB处理后，研究人员通过PCHi-C在ITGA2基因座处检测到启动子-增强子相互作用。染色质免疫共沉淀测序（ChIP-Seq）与RNA纯化染色质分离定量PCR（ChIRP-qPCR）结果显示，LncRNA-ITGA2可直接结合ITGA2增强子，并增强ITGA2增强子与ITGA2启动子区域的H3K27乙酰化修饰。此外，通过ChIRP-MS与RNA免疫沉淀实验，研究人员证实LncRNA-ITGA2可与DNA结合蛋白NONO（非POU结构域包含八聚体结合蛋白）相互作用，而ChIP-qPCR实验验证该蛋白亦可结合ITGA2启动子。最终，针对NONO、LncRNA-ITGA2及其启动子的基因敲除细胞系验证了上述调控机制。为探究血管平滑肌重构中的DNA染色质环结构，研究人员对经PDGF-BB处理与未处理的人主动脉平滑肌细胞开展了启动子捕获Hi-C实验。