The Mechanism Of IL-37-Mediated M6a RNA Methylation In Lung Adenocarcinoma

Objective Lung adenocarcinoma is one of the most common malignant tumors in the world,with a low cure rate and poor five-year survival rate.Previous studies have shown that interleukin 37?IL-37?can inhibit the proliferation,migration and invasion of lung adenocarcinoma cells,but the specific mechanism is unclear.This study explored the molecular mechanism of IL-37 inhibiting the proliferation of lung adenocarcinoma cells by regulating m6A RNA methylation,and provided a new theoretical basis for IL-37 to interfere with the biological behaviors of lung adenocarcinoma cells from the perspective of epigenetics.Methods Through analysis and in-depth mining of the TCGA database?The Cancer Genome Atlas,TCGA?and starBase database,the differences in the expression of IL-37in lung adenocarcinoma and adjacent tissues and the correlation with the prognosis of lung adenocarcinoma patients were clarified.Quantitative real-time Polymerase Chain Reaction?qRT-PCR?and Western-blot were used to investigate the expression of IL-37 in lung adenocarcinoma cell line A549 and normal lung epithelial cell line BEAS-2b.Lentivirus system was used to over expression of IL-37 in A549 cells,and their overexpression efficiency was verified.In addition,the difference in total RNA methylation before and after IL-37 overexpression was detected by colorimetry.The high-throughput sequencing and immunoprecipitation of MeRIP-Seq?Methylated RNA Immunoprecipitation sequening,MeRIP-Seq?technology and bioinformatics analysis were used to explore the basic characteristics of mRNA methylation before and after IL-37 overexpression,the distribution of m6A peaks in genes and the obvious m6A differential genes.The related biological processes and signal pathways involved in differential genes were analyzed by GO?GO ontology,GO?and KEGG?Kyoto Encyclopedia of Genes and Genomes,KEGG?.Supplemented with qRT-PCR and Western-blot to verify the effect of IL-37 on the expression of m6A RNA methylation-related protein in A549 cells.The in vivo experiment was to construct a nude mouse model of subcutaneous tumorigenesis,and intervened with IL-37 to discuss and verify the results of the above cell experiments.GraphPad Prism5 comes with statistical software for statistical analysis of the data,P<0.05 that the difference is statistically significant.Results TCGA and starBase database analysis showed that the expression of IL-37 in human lung adenocarcinoma tissues was significantly down-regulated by about 40%?t=5.62,P<0.05?,and the expression of IL-37 was positively correlated with the prognosis of patients with lung adenocarcinoma?P<0.01?.The expression of IL-37 mRNA and protein in lung adenocarcinoma A549 cells was significantly lower than that of normal lung epithelial cells BEAS-2b cells by approximately 80%and 70%,respectively(t_mRNA=14.12,P_mRNA<0.05;t_protein=9.481,P_protein<0.05).IL-37 mRNA and protein in A549 cells overexpressed IL-37 were significantly increased by about 90%and 80%(t_mRNA=9.231,P_mRNA<0.05;t_protein=9.481,P_protein<0.05).The mRNA and protein expression of demethylase ALKBH5?a-ketoglutarate-dependent dioxygenase alkB homolog 5,ALKBH5?were significantly decreased by about 30%(t_mRNA=5.014,P_mRNA<0.05;t_protein=5.740,P_protein<0.05),and the overall m6A RNA content increased significantly by about1.5 fold change?t=56.12,P<0.001?.MeRIP-Seq combined with bioinformatics analysis showed that the overall distribution of m6A before and after IL-37 overexpression was conservative.The m6A differential gene is rich in the conserved m6A motif(P=6.99e^-7).The m6A changes caused by IL-37 are related to various biological processes,such as RNA nuclear export,protein import into the nucleus,and the regulation of stem cell pluripotency.IL-37 can promote m6A methylation of Wnt5b mRNA in the upstream region of 5'UTR.The expression of Wnt5b protein in lung adenocarcinoma cell A549 was significantly increased by about 35%than BEAS-2b?t=7.551,P<0.05?,but after overexpression of IL-37,Wnt5b protein expression was significantly reduced by about60%compared with the control group?t=13.6,P<0.01?.Subcutaneous tumor formation experiments in nude mice confirmed that after overexpression of IL-37,the tumor tissue weight and volume were significantly reduced by about 60%compared with the control group?tvolume=2.704,Pvolume<0.05;tweight=2.330,Pweight<0.05?.Conclusion The expression of IL-37 in lung adenocarcinoma tissues and A549 cells was significantly lower than that in adjacent tissues and normal lung epithelial cells.It is preliminarily concluded that IL-37 promotes the methylation of overall m6A RNA,especially Wnt5b mRNA,by inhibiting the expression of ALKBH5,and then inhibits the expression of Wnt5b protein,thereby playing an important role in inhibiting the proliferation of lung adenocarcinoma cells and other biological behaviors.