The expression and function of long noncoding RNAs (lncRNAs) in the development of hypoxic pulmonary hypertension (HPH), especially in the proliferation of pulmonary artery smooth muscle cells (PASMCs), are largely unknown. Herein, we examined the expression and role of lncRNA-maternally expressed gene 3 (lncRNA-MEG3) in HPH. lncRNA-MEG3 was significantly increased and primarily localized in the cytoplasm of hypoxic PASMCs. lncRNA-MEG3 knockdown by lung-specific delivery of small interfering RNAs (siRNAs) significantly inhibited the development of HPH in vivo. Silencing of lncRNA-MEG3 by siRNAs and gapmers attenuated proliferation and cell-cycle progression in both PASMCs from idiopathic pulmonary arterial hypertension (iPAH) patients (iPAH-PASMCs) and hypoxia-exposed PASMCs in vitro. Mechanistically, we found that lncRNA-MEG3 interacts with and leads to the degradation of microRNA-328-3p (miR-328-3p), leading to upregulation of insulin-like growth factor 1 receptor (IGF1R). Additionally, higher expression of lncRNA-MEG3 and IGF1R and lower expression of miR-328-3p were observed in iPAH-PASMCs and relevant HPH models. These data provide insights into the contribution of lncRNA-MEG3 to HPH. Upregulation of lncRNA-MEG3 sequesters cytoplasmic miR-328-3p, eventually leading to expression of IGF1R, revealing a regulatory mechanism by lncRNAs in hypoxia-induced PASMC proliferation.

Background: Circular RNAs (circRNAs) have been implicated in pulmonary hypertension progression through largely unknown mechanisms. Pulmonary artery endothelial cell (PAEC) dysfunction is a hallmark in the pathogenesis of pulmonary hypertension. However, the specific role of circular RNAs in PAEC injury caused by hypoxia remains unclear. Methods: In this study, using the Western blotting, RNA pull down, Dual-luciferase reporter assay, immunohistochemistry, and immunofluorescence, we identified a novel circular RNA derived from alternative splicing of the keratin 4 gene (circKrt4). Results: CircKrt4 was upregulated in lung tissues and plasma and specifically in PAECs under hypoxic conditions. In the nucleus, circKrt4 induces endothelial-to-mesenchymal transition by interacting with the Pura (transcriptional activator protein Pur-alpha) to promote N-cadherin gene activation. In the cytoplasm, increased circKrt4 leads to mitochondrial dysfunction by inhibiting cytoplasmic-mitochondrial shuttling of mitochondrial-bound Glpk (glycerol kinase). Intriguingly, circKrt4 was identified as a super enhancer-associated circular RNA that is transcriptionally activated by a transcription factor, CEBPA (CCAAT enhancer binding protein alpha). Furthermore, RBM25 (RNA-binding-motif protein 25) was found to regulate circKrt4 cyclization by increase the back-splicing of Krt4 gene. Conclusions: These findings demonstrate that a super enhancer-associated circular RNA-circKrt4 modulates PAEC injury to promote pulmonary hypertension by targeting Pura and Glpk.

Background: Aberrant expression of circular RNAs (circRNAs) contributes to the initiation and progression of pulmonary hypertension (PH). Hypoxia‐inducible factor (HIF) is a well‐known modulator of hypoxia‐induced PH. The role and underlying mechanism of circRNAs in the regulation of HIF expression remains elusive. Methods and Results: We profiled pulmonary artery transcriptomes using RNA sequencing and screened circRNAs associated with hypoxia treatment. The expression of a novel circRNA, circ_chr11_67292179–67294612 (circ‐myh8), was increased by hypoxia in a time‐dependent manner. We evaluated the effects of circ‐myh8 overexpression by adeno‐associated virus or inhibition by short hairpin RNA on proliferation and cell cycling in mice and pulmonary artery smooth muscle cells. Overexpression of circ‐myh8 promotes PH under normoxia, and disruption of circ‐myh8 by short hairpin RNA mitigates PH in chronic hypoxic mice. Biologically, circ‐myh8 induces the proliferation and cell‐cycle progression of pulmonary artery smooth muscle cells in vivo and in vitro. Mechanistically, RNA pull‐down and RNA immunoprecipitation assays were used to examine the interaction of circRNAs with the binding protein KAT7 (lysine acetyltransferase 7). The acetylation level of lysine 5 of histone H4 in the transcriptional initiation region of HIF1α was determined by chromatin immunoprecipitation assay followed by reverse transcription‐quantitative polymerase chain reaction. Circ‐myh8 acts as a modular scaffold to recruit histone acetyltransferase KAT7 to the promoters of HIF1α, which elicits acetylation of lysine 5 of histone H4 in their promoters. Conclusions: Our findings not only reveal the pivotal roles of circ‐myh8 in governing histone modification in anti‐PH treatment but also advocate triggering the circ‐myh8/KAT7/HIF1α pathway to combat PH.

BACKGROUND: Ferroptosis has been implicated in pulmonary hypertension (PH), and chromatin-associated RNAs are increasingly recognized as key regulators of this process. However, the detailed mechanism remains unexplored. METHODS: Bioinformatics, Sanger sequencing, and RNase R digestion were used to identify the upregulation of ca-circSCN8A. Functional gain and loss assays were used to unveil the role of ca-circSCN8A in hypoxic redox–dependent ferroptosis in human pulmonary arterial smooth muscle cells and a PH mice model. Interaction between ca-circSCN8A and FUS was detected via RNA immunoprecipitation and pull-down assays. Fluorescence recovery after photobleaching, ChIRP-qPCR (Chromatin Isolation by RNA Purification followed by Quatitative PCR), malondialdehyde, reduced glutathione, and glutathione were conducted to explore the potential molecular mechanism. RESULTS: ca-circSCN8A was identified and confirmed to be upregulated in PH. Its overexpression promoted hypoxia-induced ferroptosis in human pulmonary arterial smooth muscle cells. Under hypoxic conditions, ca-circSCN8A recruited EP300 to facilitate the lactylation of FUS (Fused in Sarcoma), triggering the formation of a ca-circSCN8A/FUS/EP300 complex via liquid-liquid phase separation. Liquid-liquid phase separation maintained the stability of the R-loop formed by ca-circSCN8A and ferroptosis-related gene SLC7A11 (solute carrier family 7 member 11) promoter that inhibits its transcription, further result in the disruption of the redox homeostasis and causing ferroptosis in human pulmonary arterial smooth muscle cells. CONCLUSIONS: ca-circSCN8A recruits EP300 to promote the lactylation of FUS, thereby driving liquid-liquid phase separation–mediated complex formation with FUS and EP300. This process enables ca-circSCN8A to form an R-loop with the nonhost SLC7A11 promoter, contributing to the regulation of hypoxia-induced ferroptosis in human pulmonary arterial smooth muscle cells. This study provides the first evidence that circRNAs can form R-loops with nonhost genes in a liquid-liquid phase separation–dependent manner. Our findings highlight ca-circSCN8A as a crucial regulator of ferroptosis in hypoxic PH and a potential therapeutic target for PH.

BACKGROUND: The phenotypic transition of pulmonary artery smooth muscle cells (PASMCs) is a central pathological alteration in pulmonary artery remodeling, contributing to pulmonary hypertension. Super-enhancers (SEs), characterized by histone modifications and the binding of coactivators, drive the expression of prominent genes that define cellular identity. However, the specific role of SEs, particularly SE-driven lncRNAs (long noncoding RNAs), in hypoxia-induced phenotypic plasticity of PASMCs remains unclear. METHODS: In this study, the lncRNA UNC5B antisense RNA 1 (UNC5B-AS1) regulated by SEs was screened in hypoxic PASMCs using RNA sequencing and H3K27ac (histone 3 lysine 27 acetylation) ChIP (chromatin immunoprecipitation) sequencing. Overexpression or knockdown of UNC5B-AS1 in vitro was performed to elucidate its role in pulmonary hypertension pathogenesis. A serotype 5 adenovirus-associated virus carrying a conserved functional fragment of UNC5B-AS1 was used to treat pulmonary hypertension in vivo. RESULTS: We identified UNC5B-AS1 as an SE-driven lncRNA transcriptionally activated by the transcription factor FOXP3 (forkhead box protein P3), which regulates phenotypic transition in PASMCs. Notably, we demonstrated that UNC5B-AS1 interacts with key glycolytic enzymes in the cytoplasm and likely serves as a molecular scaffold for LRPPRC (leucine-rich PPR motif-containing protein) and oxidative respiratory chain complex IV in mitochondria. Consequently, the deficiency of UNC5B-AS1 in PASMCs promotes the lactylation of promoter regions within inflammatory genes, including those of IL (interleukin)-1β, IL-6, and TNF-α (tumor necrosis factor-α), under hypoxic conditions, ultimately leading to inflammatory phenotypic transition of PASMCs. CONCLUSIONS: Our findings identify SE-driven UNC5B-AS1 as a novel regulatory factor in the hypoxia-induced phenotypic transition of PASMCs and suggest that overexpression of UNC5B-AS1 may represent a promising therapeutic strategy for pulmonary hypertension.