Expression Of Methyltransferase Protein 14 In Intestinal Epithelial Cell Inflammation And Its Mechanism

Objective: To clarify the expression pattern of RNA N6-methyladenine(m6A)modification in the process of intestinal inflammation,to analyze the molecular regulatory mechanism of inflammation-induced m6 A modification,and to reveal the role of the key regulatory molecule methyltransferase-like protein 14(METTL14)in the inflammatory response of intestinal epithelial cells,providing new horizon for the prevention and treatment of inflammatory bowel disease(IBD).Methods:(1)The acute enteritis model of mice induced by sodium dextran sulfate(DSS)was constructed.Twenty mice were randomly divided into control group and experimental group.The experimental group was fed with 3% DSS for 7 days and sacrificed for sampling;(2)Lipopolysaccharides(LPS)-induced acute enteritis mouse model was constructed.Twenty mice were randomly divided into the control group and the experimental group.After 12 hours of fasting,10% mannitol was given to the mice by intragastric administration.After the mice were excreted,10 mg/kg LPS was injected into the rectum through the anus of the mice to induce acute colitis.(3)Dot Blot was used to analyze the changes of RNA m6 A methylation modification in the mouse enteritis model induced by two different inducers;Western blot was used to observe the changes of METTL14 and Nuclear Factor k B(NF-k B)in mice enteritis models.(4)Immunohistochemical method was used to detect the expression of METTL14 in human colitis tissue samples;(5)The toxicity of LPS and NF-KB inhibitor QNZ on NCM460 cell line was detected by CCK-8 assay.LPS and NF-KB inhibitor QNZ on NCM460 cell line.(6)the optimal concentration of LPS and QNZ was detected by Q-PCR.(7)Intestinal epithelial cell line NCM460 was induced by LPS at 1000 ng/ m L to establish in vitro intestinal epithelial cell inflammatory model;(8)Construction of METTL14 knockdown plasmid using CRISPR/Cas9 technology;(9)The expression levels of IL-1β,IL-6 and TNF-α were detected by Q-PCR in NCM460 cell line with 1000 ng/ m L LPS-induced inflammation and METTL14 overexpression and interference.(10)NF-KB inhibitor QNZ with a concentration of 1 μM was used to inhibit the expression of NF-KB in inflammatory intestinal epithelium,and the role of RNA m6 A methylation modification in the inflammatory response of intestinal epithelium was analyzed.Results:(1)In 3% DSS and 10 mg/kg LPS-induced acute enteritis mouse model,RNA m6 A methylation,METTL14 and NF-KB expression were up-regulated;(2)The expression of METTL14 was elevated in human colitis tissue samples by immunohistochemical detection;(3)The expression of m6 A,METTL14 and NF-KB were up-regulated in l PS-induced intestinal epithelial inflammation model,which was consistent with the results in mouse acute colitis model.Interference of METTL14 in NCM460 cells inhibited cell proliferation.(4)LPS was added into NCM460 cells to induce inflammation in intestinal epithelial cells,and it was found that the inflammatory response of intestinal epithelial cells after interference with METTL14 gene was significantly increased compared with the control cells.(5)The expression of m6 A and METTL14 was inhibited by the addition of NF-KB inhibitor QNZ after LPS-induced intestinal epithelial cell inflammation.Conclusion: RNA m6 A methylation is upregulated in intestinal inflammation and METTL14 may play an important regulatory role.METTL14 gene has a protective role in LPS-induced intestinal epithelial cell inflammation.Inhibition of NF-KB expression in LPS-induced intestinal epithelial inflammation down-regulates RNA m6 A methylation levels and METTL14 expression.