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中性粒细胞微囊泡调控人滑膜成纤维细胞LINE1基因甲基化

Neutrophil-derived Microvesicles Regulate DNA Methylation of the LINE1 Gene in Human Fibroblast-like Synoviocytes

    摘要
  • 摘要:
    目的 研究中性粒细胞微囊泡（neutrophil-derived microvesicles, NDMV）通过调控DNA甲基转移酶（DNA methyltransferases, DNMT）促进成纤维样滑膜细胞（fibroblast-like synoviocytes, FLS，或称滑膜成纤维细胞）LINE1基因高甲基化。
    方法 分离健康志愿者外周静脉血中的中性粒细胞并用肿瘤坏死因子α（tumor necrosis factor α, TNFα）刺激其产生NDMV。原代培养FLS至第4代备用。MTT实验检测NDMV处理FLS适宜比和时间。实验分为对照组、TNFα组、NDMV组和TNFα+NDMV组，采用硫酸氢钠联合限制性内切酶分析法（combined bisulfite restriction analysis, COBRA）检测整体和两个位点LINE1基因甲基化程度，运用RT-qPCR和Western blot检测各组磷酸化和非磷酸化DNA甲基转移酶1（DNMT1）的表达水平。
    结果 NDMV∶FLS≤100∶1时无明显细胞毒性（P>0.05）。NDMV处理FLS 24 h内对细胞活性无明显影响（P>0.05）。TNFα组LINE1基因整体甲基化程度为（37.62±2.38）%，与对照组（58.97±1.50）%、NDMV组（59.59±1.15）%和TNFα+NDMV组（59.11±0.85）%间差异均有统计学意义（P<0.001）；对照组、NDMV组和TNFα+NDMV组LINE1基因位点1和2甲基化程度分别与TNFα组比较差异亦均有统计学意义（P<0.01）。对照组、TNFα组、NDMV组和TNFα+NDMV组磷酸化DNMT1蛋白相对表达量分别为1.00±0.01、0.40±0.01、2.18±0.02和1.58±0.05（P<0.001）。
    结论 NDMV通过调控FLS的磷酸化DNMT1表达提高LINE1基因甲基化程度。

     

    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.

     

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