N6-甲基腺嘌呤去甲基酶 FTO 调节视网膜色素上皮细胞中炎症细胞因子的分泌和紧密连接。
N6-methyladenosine demethylase FTO regulates inflammatory cytokine secretion and tight junctions in retinal pigment epithelium cells.
机构信息
The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; Chongqing Key Laboratory of Ophthalmology, Chongqing, China; Chongqing Eye Institute, Chongqing, China; Chongqing Branch of National Clinical Research Center for Ocular Diseases, Chongqing, China.
College of Basic Medicine, Chongqing Medical University, Chongqing, China.
出版信息
Clin Immunol. 2022 Aug;241:109080. doi: 10.1016/j.clim.2022.109080. Epub 2022 Jul 22.
OBJECTIVE
Uveitis is an intraocular inflammatory disease. Epigenetics has been associated with its pathogenesis. However, the role of N6-methyladenosine (m6A) in uveitis has not been reported. We aimed to examine the role of m6A and its regulatory mechanism in experimental autoimmune uveitis (EAU).
METHODS
The mRNA expression of m6A-related methylase and demethylase of retinal pigment epithelium (RPE) between mice with EAU and control mice was detected by RT-qPCR. The overall m6A level of ARPE-19 cells was detected by an m6A quantitative detection kit. Cell proliferation was observed by CCK-8 assays, and ELISA was used to test the secretion of inflammatory factors. The expression of tight junction proteins and the target genes of FTO were examined by western blotting and MeRIP-PCR.
RESULTS
A decreased expression of FTO in RPE cells was found in mice with EAU. Increased overall m6A%, proliferation of cells and secretion of IL-6, IL-8 and MCP-1 were found after FTO knockdown in ARPE-19 cells. However, ZO-1 and occludin protein expression was decreased. ATF4 protein expression was decreased in the FTO knockdown (shFTO) group as compared with the control (shNC) group. In contrast, the m6A level of ATF4 was elevated, as shown by MeRIP-PCR. Functional analysis showed that p-STAT3 expression was increased in the shFTO group, and the change in occludin expression was reversed in ATF4 rescue experiment.
CONCLUSION
FTO may affect the translation of ATF4 by regulating its m6A level, resulting in the increased expression of p-STAT3 and inflammatory factors, and leading to uveitis.
目的
葡萄膜炎是一种眼内炎症性疾病。表观遗传学与其发病机制有关。然而，N6-甲基腺苷（m6A）在葡萄膜炎中的作用尚未报道。我们旨在研究 m6A 及其在实验性自身免疫性葡萄膜炎（EAU）中的调控机制的作用。
方法
通过 RT-qPCR 检测 EAU 小鼠和对照小鼠视网膜色素上皮（RPE）中 m6A 相关甲基转移酶和去甲基化酶的 mRNA 表达。通过 m6A 定量检测试剂盒检测 ARPE-19 细胞的整体 m6A 水平。通过 CCK-8 测定观察细胞增殖，ELISA 法检测炎症因子的分泌。通过 Western blot 和 MeRIP-PCR 检测紧密连接蛋白的表达和 FTO 的靶基因。
结果
在 EAU 小鼠中发现 RPE 细胞中的 FTO 表达降低。在 ARPE-19 细胞中敲低 FTO 后，发现整体 m6A%增加、细胞增殖和 IL-6、IL-8 和 MCP-1 的分泌增加。然而，ZO-1 和 occludin 蛋白表达减少。与对照（shNC）组相比，FTO 敲低（shFTO）组 ATF4 蛋白表达减少。相反，MeRIP-PCR 显示 ATF4 的 m6A 水平升高。功能分析表明，shFTO 组中 p-STAT3 表达增加，ATF4 挽救实验中 occludin 表达的变化得到逆转。
结论
FTO 可能通过调节其 m6A 水平影响 ATF4 的翻译，导致 p-STAT3 和炎症因子表达增加，从而导致葡萄膜炎。
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