PB0912 - ZC3H13-mediated m6a methylation mitigates endothelial injury through inhibiting excessive mitochondrial fission in DVT

Background: Endothelial injury, likely the core influencer of deep venous thrombosis(DVT), has long been neglected. N6-methyladenosine (m6A) is the most prevalent and abundant post-transcriptional modification on eukaryotic RNAs and, which is involved in metabolism and cellular processes. At present, the role of m6A modifications in regulating vascular endothelial injury in DVT remains elusive.

Aims: To figure out the concrete mechanisms of m6A modification in regulating endothelial injury in DVT.

Methods: M6A levels in vascular tissues were evaluated by m6A dot blot and quantification analysis. Quantitative real-time PCR, western blot, and immunofluorescence were performed to explore the expression profile of ZC3H13 in HUVECs and IVC tissues. Transmission electron microscopy, mitochondrial morphology analysis, and JC-1 staining were applied to detect mitochondrial dynamics. Effects of ZC3H13 on endothelial injury and mitochondrial fission were investigated through in vitro and in vivo experiments. Mechanism of ZC3H13-mediated m6A modification was explored through transcriptome sequencing (RNA-seq), methylated RNA immunoprecipitation sequencing (MeRIP-seq), MeRIP-qPCR, luciferase reporter assays, RNA stability assay, and RNA immunoprecipitation assay.

Results: ZC3H13 expression was significantly downregulated in injured vascular endothelium. Besides, elevated mitochondrial fission was observed in injured vascular endothelium. Functional verification has defined ZC3H13 mitigates endothelial injury through inhibiting elevated mitochondrial fission. We have revealed ZC3H13-mediated m6A modification profile in endothelial cells. Dual-specificity tyrosine-regulated kinase 1B (DYRK1B) was identified as the target gene for ZC3H13-mediated m6A modification by using RNA-seq and MeRIP-seq. Decreased m6A modification mediated by downregulation of ZC3H13 upregulated DYRK1B mRNA expression through inhibiting DYRK1B mRNA decay in a YTHDF2-dependent manner. DYRK1B was upregulated in the injured vascular endothelium. DYRK1B knockdown inhibited elevated mitochondrial fission and reversed the release of procoagulant and prothrombotic molecules in injured endothelial cells.

Conclusion(s): The findings of the current study showed that ZC3H13 mitigates endothelial injury by inhibiting excessive mitochondrial fission through the m6A/YTHDF2/DYRK1B axis.