Study On The Effect And Mechanism Of M6A Methyltransferase METTL16 On The Development Of Glioblastoma

Objective:The purpose of this study is to explore the effect of METTL16 gene on the occurrence,development and Chemotherapy Sensitivity of glioblastoma,and to reveal its potential molecular biological mechanism,in order to provide new targets and strategies for precise therapy of glioblastoma.Methods:1.Bioinformatics Analysis1.1 The differential expression of METTL16 gene in 34 kinds of tumors and peri-tumor normal tissues was analyzed by TCGAand GTEx database,and the correlation between METTL16 and prognosis of each tumor was analyzed.1.2 CGGA and GEPIA databases were used to compare the expression of METTL16 between gliomas and normal tissues,and HPA database was used to verify the results.The correlation between METTL16 and glioma WHO grade,molecular markers and sex,age,primary / recurrence were analyzed.1.3 Cancer SEA database was used to analyze the correlation between METTL16 and glioma phenotype(DNA damage / repair、cell cycle、invasion、EMT、angiogenesis、hypoxia and inflammation).2.Cell biology experiment2.1 U87 and U251 human glioblastoma cell lines(hereinafter referred to as experimental cell lines)were cultured in vitro,and METTL16 overexpression and interference vectors were constructed and transfected into experimental cell lines to obtain U87 and U251 cell lines with stable overexpression and Knockdown METTL16.According to the mode of cell intervention,the experimental cell lines were divided into Control group(blank control group),OE-NC group(overexpression negative control group),OE-METTL16 group(METTL16gene overexpression group),si-NC(knockdown negative control group)and si-METTL16group(METTL16 gene knockdown group).2.2 CCK-8 assay was used to detect the effect of overexpression / knockdown of METTL16 on the proliferation of experimental cell lines,U87 and U251 human glioblastoma TMZ resistant cell lines were induced and constructed in vitro by intermittent concentration gradient doubling method,and the effect of METTL16 overexpression on Chemotherapy Sensitivity of drug resistant experimental cell line TMZ was detected by CCK-8 assay.2.3 The effects of over-expression / knockdown of METTL16 on cell migration and invasiveness were studied by cell scratch test and Transwell chamber experiment.2.4 The effects of overexpression of METTL16 on apoptosis and cell cycle of experimental cell lines were detected by flow cytometry.2.5 Western Blot was used to detect the effect of METTL16 knockdown on the expression levels of IL-1β,NF-κB,MYD88,NLRP3,GSDMD and Caspase-1.Results:1.Searching the expression level of METTL16 in 34 kinds of tumor tissues and peri-tumor normal tissues in TCGA and GTEx database,it was found that the expression of METTL16 was different in different tumors(P<0.05).and the expression of METTL16 was significantly correlated with the prognosis of LAML、UVM、GBMLGG,、CESC and PAAD(P < 0.05).The results of GEPIA,CGGA and HPA database analysis showed that the expression level of METTL16 in glioma tissue was significantly higher than that in normal tissue samples(P<0.05),and METTL16 was significantly correlated with IDH mutation and1p/19 q deletion in low grade glioma(P<0.05).In addition,the expression level of METTL16 was not significantly correlated with the overall survival time of glioma patients(P >0.05).However,high expression of METTL16 could significantly improve the progression-free survival(P < 0.05).The correlation between METTL16 and phenotype of glioma was analyzed by Cancer SEA database.METTL16 gene may be involved in the functional processes of glioma cells,such as DNA damage / repair,cell cycle,metastasis,EMT,angiogenesis,hypoxia),inflammationand dormancy(P<0.05).2.The results of Western Blot and q PCR showed that the m RNA and protein expression levelsof the experimental cell lines transfected with overexpression vector were significantly increased(P<0.01),while the m RNAand protein expression levelsof the experimental cell lines transfected with si RNA vector were significantly decreased(P<0.05).CCK-8 and Ed U proliferation assay showed that the cell proliferation ability of si-METTL16 group was significantly higher than that of Control and NC group,while that of oe-METTL16 group was significantly inhibited(P<0.05).The results of tumor drug sensitivity test showed that the drug sensitivity of drug-resistant experimental cell lines(U87TR and U251TR)to TMZ was significantly lower than that of normal control cell lines(U87 and U251)(P<0.05),and the effect of overexpression of METTL16 on the sensitivity of drug-resistant experimental cell line TMZ was detected by CCK-8 assay.The results showed that the drug-resistant experimental cell lines in oe-METTL16 group were more sensitive to TMZ treatment than Control and oe-NC groups(P<0.01).3.Cell scratch test and transwell assay showed that in U87 and U251 cell lines,the cell migration rate and the number of invasive cells in oe-METTL16 group were significantly lower than those in Control and oe-NC groups(P<0.05).4.The results of flow cytometry showed that the apoptosis rate of experimental cell lines in oe-METTL16 group was significantly higher than that in Control and NC groups(all P <0.01).In addition,overexpression of METTL16 could induce cell cycle arrest in G2/M phase(all P < 0.01).5.Western Blot showed that the expression levels of IL-1β,NF-κB,MYD88,NLRP3,GSDMD and caspase-1 in OE-METTL16 group were significantly increased compared with those in Control and oe-NC groups.The difference was statistically significant(all P < 0.05).Conclusion:1.The differential expression of METTL16 is significantly correlated with the prognosis of GBM and other malignant tumors,and can be used as a potential molecular marker for the prognosis evaluation of GBM patients.2.METTL16 gene can effectively inhibit the proliferation,invasion and migration ability of GBM cells.In addition,overexpression of METTL16 can significantly improve the sensitivity of drug-resistant GBM cells to TMZ chemotherapy.3.METTL16 may regulate pyroptosis of glioma cells and inhibit tumor growth by activating the GSDMD signaling pathway.4.METTL16 may be involved in and induce G2/M arrest of glioma cells to inhibit tumor growth.5.METTL16 can be used as a m6 A methyltransferase with antitumor effect,providing a new target and theoretical basis for the targeted therapy of drug-resistant glioblastoma.