The Roles And Mechanisms Of M6A Demethylases FTO And ALKBH5 In Gastric Cancer

Objective: Gastric cancer(GC)is the fifth most prevalent malignant cancer and the third leading cause in tumor-related mortality worldwide.Although significant progress has been made over the past few decades,overall survival of GC is still poor.Especially,the rate of five-year survival for patients diagnosed with distant-metastasis disease remains less than 5%.Therefore,it remains essential to identify more specific biomarkers to improve early diagnosis of GC.In the field of epigenetics,N6-methyladenosine(m6A),as the most prevalent RNA methylation modification,has gradually aroused more and more attention.RNA m6 A methylation is regulated by methyltransferases(writers),demethylases(erasers)and m6A-binding proteins(readers)in a dynamic and reversible manner.Recently,accumulating studies have unveiled that aberrant m6 A modification might be involved in carcinogenesis.This study aims to investigate the roles of two demethylases FTO and ALKBH5 on the occurrence and development of gastric cancer and their specific molecular mechanisms.Methods: 1.The prognosis value of m6 A modulators on gastric cancer were analyzed using TCGA,GSE62254 and GSE15459 databases;2.The downstream targets of ALKBH5-mediated-m6 A demethylation were screened by m6 A sequencing and transcriptome sequencing;3.The expression level of mRNA and protein were examined by qRT-PCR and Western blot;4.The expression of target genes were attenuated by siRNA;5.The expression of target genes were overexpressed by plasmid;6.The ability of migration and invasion were performed by transwell assay;7.The ability to adhere to the extracellular matrix was performed by cell-extracellular matrix adhesion assay;8.The ability of cell proliferation was performed by MTT and colony formation assay;9.The enrichment of m6 A methylation modification containing mRNA was detected by methylated RNA Immunoprecipitation-qRT-PCR;10.The statistical analysis were performed by R software(V 4.0.0)and Graph Pad Prism 8.Data are shown as means ±SD obtained from three independently repeated experiments.P< 0.05 was regarded statistically significant.Results: 1.High expression of m6 A demethylase FTO predicted poor prognosis in gastric cancer.To screen out the prognostic risk factors of gastric cancer in m6 A RNA methylation modulators,the effect of 33 m6 A regulators on OS of GC was evaluated by univariate and multivariate Cox regression analysis using TCGA dataset.m6 A demethylase FTO was the most potent prognostic risk factor of gastric cancer,among all known RNA methylation regulators.2.FTO promoted GC cell migration and invasion.BGC823 and MGC803 cells were transfected with siRNAs targeted to FTO.Silencing of FTO in GC cell lines significantly decreased the ability of migration and invasion.3.Construction of weight co-expression modules with FTO expression.To clarify the molecular mechanism of FTO on GC metastasis,a gene-weighted co-expression network of FTO expression was constructed.Through cluster analysis and correlation analysis,the turquoise module was the top one positively associated with FTO expression among all ten modules.4.Function enrichment analysis of key module.The genes in the turquoise module were performed to enrichment analysis.The top 10 pathways uncovered multiple metastasis-related pathways,such as “FOCAL ADHESION(FA)”and “ECM-RECEPTOR INTERACTION”.GO enrichment analysis also revealed that the genes in turquoise module were closely associated with oncogenesis such as “cell junction organization”,“cell-matrix adhesion” and “extracellular matrix organization”.GSEA results also unveiled that high FTO expression was largely enriched in metastasis-related pathways,such as “FOCAL ADHESION(FA)” and“ECM-RECEPTOR INTERACTION”.5.Screening of FTO demethylated target genes involved in GC metastasis.26 genes overlapping in both “FOCAL ADHESION(FA)” and “ECM-RECEPTOR INTERACTION” pathways were screened as the candidates of FTO demethylated target genes using the VENNY tool.After a correlation analysis between these 26 genes and FTO,the top five genes were selected for further m6 A methylation sites prediction.The results showed that m6 A modification sites only existed in ITGB1 and LAMC1.Kaplan-Meier survival analysis indicated that patients in high expression of ITGB1 or LAMC1 group both represented shorter OS than that in low group.GSE62254 external dataset also obtained the similar results.6.Identification of ITGB1 as FTO demethylated target gene in GC cells.FTO-KD dramatically attenuated the levels of ITGB1 mRNA and protein in MGC803 and BGC823 cells,but had no apparent effect on LAMC1 expression.Me RIP-qRT-PCR showed that silencing of FTO significantly enhanced the m6 A level of ITGB1 mRNA.The expression of ITGB1 was upregulated on both mRNA and protein level after added with cycloleucine,a small molecule inhibitor of m6 A modification.ITGB1 was over-expressed in control and FTO-KD cells.ITGB1 overexpression significantly attenuated the FTO-KD-suppressed migration and invasion.7.There are controversy over the prognosis roles of ALKBH5 on gastric cancer among different datasets.In the TCGA,GSE62254 and GSE15459 datasets,univariate Cox analysis was performed on the effect of ALKBH5 on OS of gastric cancer.ALKBH5 was a protective factor in the TCGA dataset,while in GSE62254 and GSE15459 datasets,ALKBH5 was a risk factor.8.ALKBH5 promotes the migration,invasion,adhesion and proliferation of gastric cancer cells.The expression level of ALKBH5 was attenuated by siRNA and overexpressed by plasmid.Knocked down of ALKBH5 suppressed the ability of cell migration,invasion,adhesion to extracellular matrix and proliferation ability.On the contrary,the ability of cell migration,invasion,adhesion to extracellular matrix and proliferation ability were enhanced after ALKBH5 was overexpressed.9.m6 A sequencing combined with transcriptome sequencing to screen ALKBH5-mediated m6 A modification downstream target genes.ALKBH5-KD and NC samples were used to perform m6A-seq and transcriptome sequencing analysis.The conservative sequence of m6A(RRACH)was observed and the m6 A modified signal was mainly enriched around the3'-UTR and the stop codon.After knockdown of ALKBH5,“the Hippo signaling pathway” ranked first among the pathways in which the m6 A modification level was upregulated.And 4492 genes were down-regulated and 2409 genes were up-regulated in mRNA expression level.CTGF,AJUBA,and FZD4 overlapping in both “the Hippo signaling pathway” and down-regulated by ALKBH5 were screened as the candidates using the VENNY tool.High expression of FZD4 predicted poor prognosis in gastric cancer,while CTGF and AJUBA were not.In the GSE62254 dataset,high expression of FZD4 was significantly related to age(P=0.0015),tumor size(P<0.001),lymph node metastasis(P=0.022)and distant metastasis(P=0.007).The expression of FZD4 and ALKBH5 was positively correlated(R = 0.3153,P <0.0001).10.ALKBH5 increased the FZD4 expression by reducing its m6 A modification level.ALKBH5-KD dramatically attenuated the mRNA expression of FZD4,while overexpression of ALKBH5 upregulated the mRNA expression of FZD4.The expression level of m6A-binding protein YTHDF2 was decreased by siRNA and the expression of FZD4 was significantly up-regulated after knockdown of YTHDF2.11.FZD4 promotes the migration,invasion,adhesion and proliferation of gastric cancer cells.The expression level of FZD4 was down-regulated by siRNA.Knockdown of FZD4 suppressed the ability of cell migration,invasion,adhesion to extracellular matrix and proliferation.Conclusion: 1.m6 A demethylase FTO was the most potent prognostic risk factor of GC.2.FTO could promote GC metastasis by upregulating the expression of Integrin?1(ITGB1)via decreasing its m6 A level.3.m6 A demethylase ALKBH5 was an independent risk factor affecting the prognosis of GC.4.ALKBH5 could upregulate FZD4 expression to promote the occurrence and metastasis of GC by decreasing the m6 A level of FZD4 mRNA,which repressed the mRNA degradation function of m6 A binding protein YTHDF2.