Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response in Fusarium solani-Induced Keratitis via the NF-κB Signaling Pathway

PURPOSE. The purpose of this study was to elucidate the effect of methyltransferase-like enzyme 3 (METTL3) on inflammation and the NF-kappa B signaling pathway in fungal keratitis (FK). METHODS. We established corneal stromal cell models and FK mouse models by incubation with Fusarium solani. The overall RNA N6-methyladenosine (m6A) level was determined using an m6A RNA methylation assay kit. The expression of METTL3 was quantified via real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Subsequently, the level of tumor necrosis factor (TNF) receptorassociated factor 6 (TRAF6) was identified by Western blotting and immunofluorescence. Moreover, we assessed the effect of METTL3 by transfecting cells with siRNA (in vitro) or adeno-associated virus (in vivo). Hematoxylin and eosin (H&E) staining and slit-lamp biomicroscopy were performed to evaluate corneal damage. Furthermore, the state of NF-kappa B signaling pathway activation was examined by Western blotting. In addition, RT-PCR and enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate levels of the pro-inflammatory factors interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and TNF-alpha. RESULTS. Our data demonstrated that the levels of the RNA m6A methylation and METTL3 were dramatically increased and that the NF-kappa B signaling pathway was activated in Fusarium solani-induced keratitis. Inhibition of METTL3 decreased the level of TRAF6, downregulated the phospho-p65(p-p65)/p65 and phospho-I kappa B(p-I kappa B)/I kappa B protein ratios, simultaneously attenuating the inflammatory response and fungal burden in FK. CONCLUSIONS. Our research suggests that the m6A methyltransferase METTL3 regulates the inflammatory response in FK by modulating the NF-kappa B signaling pathway.

Automated summary

This study elucidated the effect of METTL3 on inflammation and the NF-kappa B signaling pathway in fungal keratitis. Inhibition of METTL3 decreased TRAF6 levels and attenuated the inflammatory response and fungal burden in FK through regulating the NF-κB signaling pathway.