Department of Ophthalmology, Fujian Medical University Union Hospital, Fu Zhou, China.
Invest Ophthalmol Vis Sci. 2021 Jan 4;62(1):24. doi: 10.1167/iovs.62.1.24.
Accumulated evidence has shown that microRNAs (miRNAs) are closely related with the regulation of autophagy, which plays vital roles in fungal keratitis (FK). Microarray data showed elevated expression of miR-665-3p in mouse corneal tissues after infection with Fusarium solani (F. solani). Here, we investigated the effect of miR-665-3p in regulating autophagy in experimental F. solani keratitis and determined the potential molecular mechanisms involved.
In this article, we established an in vivo mouse model of FK and an in vitro model of corneal stromal cells by inoculating with F. solani. We divided them into the following six groups: control, chloroquine (CQ), rapamycin (Rapa), miR-665-3p antagomir (ant-665), miR-665-3p agomir (miR-665), and the negative control group (miR-NC). The levels of autophagy were detected by electron microscopy, Western blotting, and immunofluorescence. Then, we used a dual-luciferase reporter assay to determine the binding of miR-665-3p to the autophagy-related gene (ATG)5 3'UTR. Detection of IL-1β protein levels and hematoxylin and eosin (H&E) staining of corneal tissues were used to observe the effect of miR-665-3p on inflammation in FK.
Here, we showed that inhibition of miR-665-3p expression in FK upregulated autophagy and alleviated inflammation. Nevertheless, the opposite results were found by overexpressing miR-665-3p. Additionally, ATG5 was a direct target gene for miR-665-3p.
Together, our data demonstrated that miR-665-3p might be involved in F. solani keratitis of mice by regulating autophagic pathways and inflammation.
已有大量证据表明，微小 RNA（miRNA）与自噬的调节密切相关，自噬在真菌性角膜炎（FK）中起着至关重要的作用。微阵列数据显示，感染茄病镰刀菌（F. solani）后，小鼠角膜组织中 miR-665-3p 的表达升高。在此，我们研究了 miR-665-3p 调节实验性 F. solani 角膜炎中自噬的作用，并确定了涉及的潜在分子机制。
在本文中，我们通过接种 F. solani 建立了 FK 的体内小鼠模型和角膜基质细胞的体外模型。我们将它们分为以下六组：对照组、氯喹（CQ）组、雷帕霉素（Rapa）组、miR-665-3p 反义寡核苷酸（ant-665）组、miR-665-3p 激动剂（miR-665）组和阴性对照组（miR-NC）。通过电子显微镜、Western blot 和免疫荧光检测自噬水平。然后，我们使用双荧光素酶报告基因检测来确定 miR-665-3p 与自噬相关基因（ATG）5 3'UTR 的结合。检测 IL-1β 蛋白水平和角膜组织的苏木精和伊红（H&E）染色用于观察 miR-665-3p 对 FK 中炎症的影响。
在这里，我们表明 FK 中 miR-665-3p 表达的抑制上调了自噬并减轻了炎症。然而，过表达 miR-665-3p 则得到了相反的结果。此外，ATG5 是 miR-665-3p 的直接靶基因。
综上所述，我们的数据表明，miR-665-3p 可能通过调节自噬途径和炎症参与了小鼠 F. solani 角膜炎。
