N6-methyladenosine demethylase FTO regulates inflammatory cytokine secretion and tight junctions in retinal pigment epithelium cells

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.