The Mechanism Of METTL3 On Regulating UC Via Activating NF-κB Signal Pathway

Objective:To investigate the expression of m6A methyltransferase like 3(METTL3)in UC and the mechanism of METTL3 on regulating UC via activating NF-κB signal pathway.Methods:1.We selected the key enzymes that had significant differences in the expression of m6A methylation between UC and normal samples by querying the GEO database,and detected the differential expression of this key enzyme in diseased colon tissues and relatively normal colon tissues of UC patients.2.We completed the establishment and identify of DSS-induced colitis model in mice,and detected the expression of METTL3 in the DSS-induced UC model in mice.3.We used lentivirus transfection technique to knock down METTL3 in LPS-induced mouse intestinal epithelial cells(MODE-K cells).The qRT-PCR and Western Blot were used to detect knockdown efficiency,and then CCK-8,flow cytometry and WB were used to evaluate the effect of METTL3 knockdown on intestinal epithelial cell inflammation induced by LPS.4.The expression of NF-κB signaling pathway before and after METTL3 knockdown was detected in LPS-stimulated MODE-K cells,and the effect of inhibiting NF-κB signaling pathway on the expression level of METTL3 was evaluated separately.5.We assigned mice into four groups:control,DSS-induced UC model,DSS-induced mice received the sh-NC injection,and DSS-induced mice received the sh-METTL3 injection.The effects of METTL3 overexpression on DSS-induced UC and the NF-κB signaling were investigated in vivo.The METTL3 inhibitor STM2457 was used to further confirm the effect of METTL3 on the UC mouse model.Results:1.The expression of METTL3 was significantly up-regulated in UC samples according to GSE87466 and GSE75214.PCR results showed that the expression of METTL3 in UC diseased colon tissues was significantly higher than that in the normal control group.2.METTL3 was upregulated in the DSS-induced UC model in mice.3.The expression of various inflammatory factors was downregulated and the apoptosis rate was decreased after METTL3 knockdown,which relives LPS-induced cellular inflammation.4.The expression of NF-κB signaling pathway was downregulated after METTL3 knockdown in LPS-stimulated MODE-K cells,and METTL3 overexpression also could be reversed by NF-κB inhibitor treatment.5.METTL3 overexpression aggravates LPS-induced cellular inflammation in mouse intestinal epithelial cells,the effects of METTL3 overexpression upon LPS-stimulated MODE-K cells were partially abolished by NF-κB inhibitor JSH-23.Similarly,METTL3 knockdown in DSS-induced UC mice also inhibited p65 phosphorylation,suggesting METTL3 regulation of the NF-κB pathway in vivo.Moreover,in the DSS-induced UC mouse model,METTL3 knockdown partially ameliorated DSS-induced intestinal injury and inflammation,as manifested as increased body weight and decreased DAI scores,decreased the expression of various inflammatory factors.The effect of STM2457 on DSSinduced UC model in mice was similar to that of METTL3 knockdown.Conclusion:The expression of METTL3 was upregulated in the clinical specimens of UC.METTL3 knockdown can inhibit the phosphorylation of NF-κB pathway protein p65 in vivo and in vitro,and then inhibit the development of UC.