Enoxaparin alleviates preeclampsia by enhancing trophoblast function via the NSUN2-mediated m(5)C methylation of PAX3 mRNA.

OBJECTIVE: Preeclampsia (PE), a complex multisystem disorder, is a major contributor to maternal and perinatal morbidity and mortality. Enoxaparin, a low-molecular-weight heparin, has shown therapeutic efficacy in preventing various diseases. This study aimed to investigate the role of enoxaparin in PE and clarify its underlying mechanisms. METHODS: Trophoblast (HTR-8/SVneo) cell viability, proliferation, migration, and invasion under hypoxia were assessed by Cell Counting Kit-8 (CCK-8), EdU incorporation, wound healing, and Transwell assays. Global RNA m(5)C levels were measured via dot blot. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was employed to quantify mRNA levels of RNA methyltransferases. The interaction between NOL1/NOP2/SUN domain-containing protein 2 (NSUN2) and paired box 3 (PAX3) was examined via RNA immunoprecipitation (RIP) assays. Additionally, 5-methylcytosine (m(5)C) sites in PAX3 were analyzed using a dual-luciferase reporter assay. A PE mouse model was established to validate findings. RESULTS: Enoxaparin enhanced trophoblast proliferation, migration, and invasion under hypoxia. It upregulated the m(5)C methyltransferase NSUN2 and increased global RNA m(5)C levels. Clinically, NSUN2 and global m(5)C levels were reduced in PE placentas. NSUN2 knockdown suppressed trophoblast function and abolished enoxaparin's benefits. Mechanistically, NSUN2 bound to PAX3 mRNA and installed m(5)C methylation, which stabilized PAX3 transcripts and promoted its expression. PAX3 overexpression rescued the impairments caused by NSUN2 knockdown. In mice, enoxaparin ameliorated hypertension, proteinuria, placental injury, and elevated fms-like tyrosine kinase-1 (sFLT-1), effects reversed by NSUN2 silencing. CONCLUSIONS: Enoxaparin mitigated PE progression by upregulating NSUN2, which enhanced PAX3 expression via site-specific m(5)C methylation, thereby improving trophoblast function. The NSUN2/m(5)C/PAX3 axis represents a novel mechanistic insight and potential therapeutic target for PE.