miRNAs介导的自噬抑制在角膜真菌感染免疫逃逸中的作用机制研究

Autophagy play an essential role in the host innate immune against pathogenic. MicroRNAs (miRNAs), as a significant signal molecule in the interactions between pathogen and host, media autophagy to eradicate pathogens. Our prior studies have indicated, that autophagy was downregulated following infection of fungal in cornea, furthermore three kind of miRNAs were found to be upregulated and targeted to the ATG10 gene which play a key role in autophagy. We can speculated that the downregulation of autophagy which mediated by miRNAs might plays an important role in the development of the fungal keratitis. In this subject, the in vitro and in vivo model of fungal keratitis were used to observe the effect of autophagy on fungal inflamation, and the expression of miRNA, which targeted to ATG10 and was supressed by the autophagy, was invesigated. Meanwhile, the research also refered to the molecule mechanism of transcription regulation. Also, by this subject, the relationships among fungus, miRNAs, autophagy and immune were revealed. The network for strategies aimed at combating fungal keratitis was also investigated and may provide useful information for developing potential therapeutic interventions against pathogens.

细胞自噬在清除病原感染中发挥重要作用；微小RNA（miRNA）作为病原与宿主相互作用的重要信号调节分子，介导了清除病原体的自噬反应。项目组前期研究发现，角膜感染真菌后细胞自噬水平显著下降，同时伴有3种miRNAs的表达显著增强，且此3种miRNAs均靶向自噬信号通路的关键分子自噬相关基因10（ATG10）。由此推测miRNAs介导的细胞自噬的下降可能在真菌性角膜炎的发生发展中具有重要作用。本课题拟通过体外与体内真菌感染角膜的模型，观察真菌感染角膜后对细胞自噬的影响，研究miRNAs通过ATG10介导自噬抑制及其转录调控的分子机制，探讨“真菌、miRNAs、自噬与免疫”的作用模式，以期阐明miRNAs介导的自噬抑制在角膜真菌感染免疫逃逸中的分子机制，进一步从机体免疫学角度研究真菌性角膜炎的发病机制和治疗策略。

机体免疫在真菌性角膜炎的病理生理过程中发挥着重要作用，但真菌感染角膜后如何逃逸机体的免疫效应以保护自己这一问题仍不清楚。研究表明，细胞自噬在清除真菌等病原感染中发挥重要作用，miRNA介导了清除病原体的自噬反应，在调节宿主抗感染免疫应答中发挥重要作用。本课题应用体外细胞培养技术及建立小鼠真菌性角膜炎模型，检测感染角膜中miRNA的表达、自噬的水平及自噬流的阻滞情况，并研究这些miRNA对角膜基质细胞的自噬抑制作用，探讨自噬通路中的信号蛋白、自噬的增强剂及miRNA的调节剂等对真菌的清除和角膜免疫反应的影响。研究发现：1.在茄病镰刀菌性角膜炎病变角膜中出现多种miRNAs（如miR-129-5p、miR-223-3p、miR-665-3p等）的表达上调，并与炎症反应密切相关；2.真菌感染的角膜组织中自噬受阻，表现为溶酶体和自噬小体融合障碍，P62上升，LC3B II/I，Beclin 1下降等；3.自噬激动剂雷帕霉素及miRNAs（如miR-129-5p、miR-223-3p、miR-665-3p等）的拮抗剂antagomir均可以促进自噬水平的上升，增强真菌孢子的清除；可抑制炎症因子的表达，减轻角膜组织炎症反应，并显著提高角膜基质细胞的活力；4.通过甲基化芯片检测，真菌感染后角膜m6A甲基化水平升高，METTL3的表达明显增强；高甲基化修饰的基因显著富集到PI3K-AKT信号通路与NF-κB 信号通路；5.通过干扰METTL3的表达发现，METTL3表达的下调能抑制PI3K/AKT和NF-κB通路的激活，从而降低致炎因子的表达，减轻角膜炎症，并帮助机体更快地清除角膜中的真菌。本研究进一步阐明miRNA介导的自噬抑制在角膜真菌感染免疫逃逸中的分子机制,从机体免疫学和m6A表观遗传调控角度研究真菌性角膜炎的发病机制,为寻找其有效的治疗药靶提供新的思路和方法。