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

The proliferation and migration of VSMCs significantly contribute to vascular remodeling. Recent studies have suggested that enhancer-associated long noncoding RNAs (elncRNAs) play crucial roles in regulating gene expression and cell fate. However, the specific elncRNAs implicated in VSMC dysfunction and their regulatory mechanisms remain poorly understood. This study used multiomics profiling, including CUT&Tag, promoter capture Hi-C (PCHi-C), and microarray analysis, to identify LncRNA-ITGA2 as a novel elncRNA highly expressed in PDGF-induced proliferative human VSMCs. Notably, LncRNA-ITGA2 was significantly elevated in the coronary atherosclerotic tissues of patients with CADcompared with those of control subjects. Gain- and loss-of-function studies suggested that LncRNA-ITGA2, markedly enhanced PDGF-induced VSMC proliferation and migration. In vivo, the overexpression of LncRNA-ITGA2 promoted neointimal hyperplasia in a mouse carotid artery injury model, indicating its partial functional conservation. Using RNA sequencing and CRISPR-Cas9 gene-editing technology, we identified integrin alpha2 (ITGA2) as a downstream target of LncRNA-ITGA2. Mechanistically, PCHi-C detected promoter-enhancer interactions at the ITGA2 locus following PDGF-BB treatment. ChIP-Seq and ChIRP-qPCR demonstrated that LncRNA-ITGA2 directly bound to the Enhancer-ITGA2 and increased H3K27 acetylation in both the Enhancer-ITGA2 and the Promoter-ITGA2. Additionally, ChIRP-MS and RNA immunoprecipitation revealed that LncRNA-ITGA2 interacted with the DNA-binding protein NONO , which also bound to the ITGA2 promoter, as confirmed by ChIP-qPCR. Ultimately, the use of knockout cell lines for NONO, LncRNA-ITGA2, and its promoter validated the proposed regulatory mechanism. This study identifies a novel mechanism by which elncRNA (LncRNA-ITGA2), in a NONO-dependent manner, mediates interactions between Enhancer-ITGA2 and Promoter-ITGA2, resulting in increased ITGA2 expression and subsequently promoting VSMC proliferation and migration. These findings highlight LncRNA-ITGA2 as an attractive diagnostic and therapeutic target for human proliferative vascular diseases