背景:YTHDC1,一个键m(6)一个核阅读器,在调节mRNA剪接中起着至关重要的作用,export,和稳定性。然而,YTHDC1在炎症性肠病(IBD)中的功能意义和调控机制仍有待探索。
方法:我们建立了葡聚糖硫酸钠(DSS)诱导的小鼠结肠炎模型,并在体外建立了LPS/IFN-γ刺激的巨噬细胞炎症。测定YTHDC1的表达。通过免疫荧光染色测定YTHDC1和巨噬细胞的共定位。应用LV-YTHDC1或shYTHDC1慢病毒用于YTHDC1过表达或抑制。对于NF-κB抑制,使用了JSH-23。通过RIP测定YTHDC1和Beclin1mRNA的相互作用,MeRIP证实了Beclin1的m6A修饰。
结果:在DSS诱导的结肠炎和LPS/IFN-γ处理的RAW264.7巨噬细胞中,我们观察到YTHDC1显著下调。YTHDC1的过表达导致iNOS水平降低,CD86和IL-6mRNA,同时抑制LPS/IFN-γ处理的RAW264.7细胞中NF-κB的活化。相反,YTHDC1下调可促进iNOS表达并抑制自噬。此外,NF-κB抑制剂JSH-23消除了YTHDC1对LPS/IFN-γ诱导的CD86和IL-6mRNA的影响。机械上,YTHDC1与Beclin1mRNA相互作用,从而稳定Beclin1mRNA并增强Beclin1表达和自噬。这些作用最终导致LPS/IFN-γ攻击的巨噬细胞中NF-κB信号的抑制。
结论:YTHDC1通过稳定Beclin1mRNA抑制巨噬细胞介导的炎症反应,这可能是治疗IBD的潜在治疗靶点。
BACKGROUND: YTHDC1, a key m(6)A nuclear reader, plays a crucial role in regulating mRNA splicing, export, and stability. However, the functional significance and regulatory mechanisms of YTHDC1 in inflammatory bowel disease (IBD) remain to be explored.
METHODS: We established a dextran sulfate sodium (DSS)-induced murine colitis model in vivo and LPS/IFN-γ-stimulated macrophage inflammation in vitro. The expression of YTHDC1 was determined. Colocalization of YTHDC1 and macrophages was assayed by immunofluorescence staining. LV-YTHDC1 or shYTHDC1 lentiviruses were applied for YTHDC1 overexpression or inhibition. For NF-κB inhibition, JSH-23 was utilized. The interaction of YTHDC1 and
Beclin1 mRNA was determined by RIP, and the m6A modification of
Beclin1 was confirmed by MeRIP.
RESULTS: In DSS-induced colitis and LPS/IFN-γ-treated RAW264.7 macrophages, we observed a significant downregulation of YTHDC1. Overexpression of YTHDC1 resulted in decreased levels of iNOS, CD86, and IL-6 mRNA, along with inhibited NF-κB activation in LPS/IFN-γ-treated RAW264.7 cells. Conversely, downregulation of YTHDC1 promoted iNOS expression and inhibited autophagy. Additionally, the effect of YTHDC1 knockdown on CD86 and IL-6 mRNA induced by LPS/IFN-γ was abolished by the NF-κB inhibitor JSH-23. Mechanistically, YTHDC1 interacted with
Beclin1 mRNA, thereby stabilizing
Beclin1 mRNA and enhancing
Beclin1 expression and autophagy. These effects ultimately led to the inhibition of NF-κB signaling in LPS/IFN-γ-challenged macrophages.
CONCLUSIONS: YTHDC1 inhibited the macrophage-mediated inflammatory response by stabilizing
Beclin1 mRNA, which may be a potential therapeutic target for the treatment of IBD.