Super-enhancer-driven lncRNA HCG20 regulates pulmonary endothelial cell dysfunction through U2AF2-mediated pre-mRNA alternative splicing
Background: Pulmonary artery endothelial cell (PAEC) dysfunction is a pathological hallmark of pulmonary hypertension (PH). Yet, the roles of long noncoding RNAs (lncRNAs) driven by super-enhancers (SE-lncRNAs) in PAECs are not well understood. In this study, we focused on the PAEC-specific SE-lncRNA HCG20 and to elucidate its role and underlying mechanisms in the progression of PH.Methods: ChIP-qPCR, 3C-PCR, CRISPR/Cas9, and dual luciferase reporter assays were employed to identify dysregulated SE-lncRNAs in PAECs and to investigate the pathological role of HCG20. The role of HCG20 in pathological processes was validated in rodent models of PH induced by SU5416/hypoxia (SuHx), monocrotaline (MCT) or hypoxia alone, through adeno-associated virus (AAV)-mediated endothelial-specific HCG20 overexpression or knockdown of HCG20. RNA pull-down, mass spectrometry, RNA immunoprecipitation and RNA-seq were employed to elucidate the underlying mechanisms of HCG20-mediated PAEC dysfunction.Results: We identified the SE-LncRNA HCG20 from H3K27ac and H3K4me1 ChIP-seq data derived from PAEC of patients with PH. A significant upregulation of the HCG20 was found in hypoxia-induced hPAECs, lung tissues and the plasma of patients with PH. Antisense oligonucleotide (ASO) and CRISPR/Cas9, which respectively target HCG20 and its SE, alleviate hypoxia-induced pyroptosis and subsequent endothelial-to-mesenchymal transition (EndMT). Human pulmonary artery smooth muscle cells (hPASMCs) internalize hPAECs-derived exosomes containing HCG20, inducing their excessive proliferation. Targeted delivery of HCG20 into the pulmonary vascular endothelium induced pulmonary vasculature remodeling and increased pulmonary artery systolic blood pressure in rodents. Mechanistically, HCG20 directly bound and stabilized the U2 small nuclear RNA auxiliary factor 2 (U2AF2) protein, thereby facilitating its impact on the alternative splicing of Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2). Furthermore, we identified a novel mouse ortholog gene, 4833427F10Rik (named Hcg20), of HCG20 for the first time. Our study demonstrated that specific interference with Hcg20 in the pulmonary vascular intima has been shown to ameliorate hypoxia-induced PH.Conclusions: Collectively, our data suggest that HCG20, driven by SE, contributes to PAEC dysfunction through U2AF2-mediated alternative splicing of EIF2AK2. Our work underscores the potential of using HCG20 as a novel biomarker and a promising target for the treatment of PH.