ca-circSCN8A promotes HPASMCs ferroptosis via LLPS initiated R-Loop

BACKGROUNDFerroptosis has been implicated in pulmonary hypertension. Chromatin-associated RNAs are linked to ferroptosis. However, their role in pulmonary vascular ferroptosis in PH is unexplored. METHODSBioinformatics, Sanger sequencing, RNase R and others were employed to identify differentially higher expression of ca-circSCN8A. Functional gain and loss assays were employed to unveil the role of ca-circSCN8A in hypoxia-induced redox-dependent ferroptosis in HPASMCs and PH mice model. Interaction between ca-circSCN8A and FUS was detected via RNA immunoprecipitation and pull-down assays. FRAP, CHIRP-qPCR, MDA, GSH and GSSG were conducted to explore the potential molecular mechanism. RESULTSca-circSCN8A was firstly identified and confirmed to be upregulated in PH. Upregulation of ca-circSCN8A can promote hypoxia induced ferroptosis in HPASMCs. Under hypoxic conditions, ca-circSCN8A promoted the lactylation of FUS by recruiting EP300, leading to formation of LLPS of ca-circSCN8A/FUS/EP300. LLPS maintained the stability of the R-Loop formed by ca-circSCN8A and ferroptosis related-gene SLC7A11 promoter that inhibit the transcription of the SLC7A11 gene, further result in the disruption of the redox balance and causes ferroptosis in HPASMCs. CONCLUSIONSca-circSCN8A recruits EP300 to promote the lactylation of FUS, thereby promoting LLPS formation of ca-circSCN8A/EP300/FUS. Further, the LLPS motivates ca-circSCN8A to form R-loop with its non-host SLC7A11 and participating in the regulation of hypoxic induced HPASMCs redox-dependent ferroptosis. This is the first confirmation that circRNAs forming R-loops with non-host genes regulated by LLPS. Our findings indicated that ca-circSCN8A plays an important role in ferroptosis of hypoxic induced HPASMCs and may be a potential target for the treatment of PH.