Abstract
OBJECTIVE: To investigate how neutrophil-derived microvesicles (NDMV) promote LINE1 gene hypermethylation in fibroblast-like synoviocytes (FLS) by regulating DNA methyltransferases (DNMT). METHODS: Neutrophils were isolated from the peripheral blood of healthy volunteers and stimulated with tumor necrosis factor α (TNFα) to produce NDMV. Primary FLS were cultured to the fourth passage for use. MTT assays determined the optimal NDMV treatment ratios and durations for FLS. Experiments groups included control, TNFα, NDMV, and TNFα + NDMV. Combined bisulfite restriction analysis (COBRA) assessed global LINE1 methylation levels and methylation at two specific sites. RT-qPCR and Western blot analyzed phosphorylated and non-phosphorylated DNA methyltransferase 1 (DNMT1) expression in each group. RESULTS: No significant cytotoxicity was observed at NDMV∶FLS ratios ≤ 100∶1 (P > 0.05). NDMV treatment for 24 hours had no significant effect on FLS cell viability (P > 0.05). The overall methylation level of the LINE1 gene in the TNFα group was (37.62 ± 2.38)%, which differed significantly from the control group (58.97 ± 1.50)%, NDMV group (59.59 ± 1.15)%, and TNFα + NDMV group (59.11 ± 0.85)%, P < 0.001. Compared with the TNFα group, methylation levels at LINE1 sites 1 and 2 in the control, NDMV, and TNFα + NDMV groups also showed statistically significant differences (P < 0.01). The relative expression levels of phosphorylated DNMT1 protein in the control group, TNFα group, NDMV group, and TNFα + NDMV group were 1.00 ± 0.01, 0.40 ± 0.01, 2.18 ± 0.02, and 1.58 ± 0.05, respectively (P < 0.001). CONCLUSION: NDMV enhances LINE1 gene methylation by regulating phosphorylated DNMT1 expression in FLS.