SRSF4-Associated ca-circFOXP1 Regulates Hypoxia-Induced PASMC Proliferation by the Formation of R Loop With Host Gene

BACKGROUND: Pulmonary hypertension (PH) is a rare and fatal disease, the pathological changes of which include pulmonary arterial smooth muscle cell (PASMC) proliferation, which is the pathological basis of pulmonary vascular remodeling. Studies have demonstrated that chromatin-associated circRNA can regulate a variety of biological processes, including PASMC proliferation in patients with hypoxic PH. However, the role of chromatin-associated circRNA in the proliferation of PH remains largely unexplored. In this study, we aimed to identify the function and mechanism of chromatin-associated circRNA in PASMC proliferation in PH. METHODS: The role of ca-circFOXP1 was investigated in hypoxic mouse PASMCs and SuHX (Sugen5416+hypoxia) model mice through the use of antisense oligonucleotide knockdown and adeno-associated virus–mediated knockdown. Through bioinformatic sequence alignment, chromatin isolation by RNA purification, Cell Counting Kit 8, 5-ethynyl-2-deoxyuridine, Western blot, and other experiments, the function and mechanism of ca-circFOXP1 were verified. RESULTS: The expression of ca-circFOXP1 was found to be significantly increased in SuHX model mice and hypoxic mouse PASMCs. Moreover, ca-circFOXP1 was found to regulate the level of the host protein FOXP1 (forkhead box protein 1) through the R loop, thereby influencing the phosphorylation activity of SMAD2 (SMAD family member 2) and, consequently, the proliferation of mouse PASMCs. It is noteworthy that the m6A modification was found to promote the formation of the R loop between ca-circFOXP1 and the host gene FOXP1 , thereby regulating the expression of the host protein. Furthermore, we have identified that the splicing factor SRSF4 (serine/arginine rich splicing factor 4) can upregulate the expression of ca-circFOXP1 by splicing exons 6 and 9 of FOXP1 pre-mRNA. CONCLUSIONS: The results demonstrated that the splicing factor SRSF4 upregulated the expression of ca-circFOXP1, and m6A methylation promoted R-loop formation between ca-circFOXP1 and host genes, regulated the level of host protein FOXP1, and then affected the phosphorylation activity of SMAD2, mediating PASMC proliferation, leading to pulmonary vascular remodeling. These results provide a theoretical basis for further study of the pathological mechanisms of hypoxic PH and may provide certain insights.