Zinc finger CCCH-type containing 13-mediated N6-methyladenosine methylation mitigates endothelial injury in venous thrombosis by inhibiting elevated mitochondrial fission.

Abstract

BACKGROUND: Endothelial injury is the core factor of venous thrombosis. N6-methyladenosine (m6A) plays a critical role in metabolism and cellular processes. Moreover, the balance of mitochondrial dynamics is essential in regulating cellular growth, apoptosis, and mobility. However, the roles of m6A modification and mitochondrial dynamics in regulating venous endothelial cells remains elusive. METHODS: Levels of m6A were evaluated by m6A dot blot and quantification analysis. Gain- and loss-of-function and rescue assays were performed to clarify gene functions. To investigate the mitochondrial dynamics in venous endothelium, mitochondrial morphology and function analysis were performed. The target gene of zinc finger CCCH-type containing 13 (ZC3H13) was identified through RNA-sequencing and methylated RNA immunoprecipitation sequencing. Mechanism of ZC3H13-mediated m6A modification was explored through methylated RNA immunoprecipitation quantitative polymerase chain reaction (qPCR), luciferase reporter assay, RNA stability assay, and RNA immunoprecipitation assay. RESULTS: Downregulated ZC3H13 expression and elevated mitochondrial fission were observed in injured venous endothelium. Functional verification has clarified ZC3H13 regulated endothelial cells by modulating mitochondrial fission. Furthermore, ZC3H13-mediated m6A modification profile was revealed, and DYRK1B was identified as its target in endothelial cells. Decreased m6A modification mediated by downregulation of ZC3H13 upregulated DYRK1B messenger RNA expression through inhibiting DYRK1B messenger RNA decay in an YTHDF-2-dependent manner. Functional verification also confirmed the functions of DYRK1B in regulating endothelial cells by modulating mitochondrial fission. Moreover, endothelial-targeted ZC3H13 overexpression attenuated venous endothelial injury, which contributed to the reduced thrombotic risk observed in mice. CONCLUSION: The findings of the current study showed that ZC3H13 mitigates endothelial injury by inhibiting excessive mitochondrial fission through the m6A/YTHDF2/DYRK1B axis.