Effect Of M6A Methyltransferase WTAP On Esophageal Squamous Cell Carcinoma And Its Mechanism

Background: Esophageal cancer(EC)is one of the aggressive gastrointestinal malignancies that causes a large number of deaths worldwide.N6-methyladenosine(m6A)modification is the most abundant modification on eukaryotic RNA.In recent years,lots of studies have reported that m6 A modification and m6 A RNA methylation regulators were involved in cancer progression.However,the m6 A level and its regulators in esophageal cancer(ESCA)remain poorly understood.Objectives: Key m6 A regulatory factors related to the pathogenesis of esophageal cancer were screened,and the expression of key m6 A regulatory factor WTAP in esophageal cancer was determined.The correlation between the expression of WTAP and the clinical data and prognosis of esophageal cancer patients was analyzed.Cytological experiments were performed to determine the effect of WTAP on proliferation,migration and invasion of esophageal carcinoma cells.The molecular mechanism by which WTAP and binding proteins regulate m6 A modification of downstream genes was preliminarily elucidated.Methods:The expression of esophageal cancer was downloaded from TCGA database and R language was used to screen the key regulatory factor m6 A differentially expressed in esophageal cancer and paracancer tissues.Oncolnc tools were used to determine the m6 A regulatory factors associated with the prognosis of esophageal cancer patients.In this study,87 cases of esophageal squamous cell carcinoma and 44 cases of normal esophageal tissue were used as research objects,and the expression of WTAP protein in esophageal squamous cell carcinoma was detected by immunohistochemical method.The relationship between WTAP protein expression and clinicopathological features and prognosis of patients was analyzed.The effects of WTAP expression on proliferation,migration and invasion of esophageal squamous cell carcinoma cells were investigated by CCK8,colony formation,scratch test and invasion test.Meanwhile,Westren Blot and RT-q PCR were used to verify the effect of WTAP on the expression of dryness related genes in tumor cells.Differentially expressed m RNA in WTAP-knocked down esophageal squamous cell carcinoma cells were screened by gene chip technique.On this basis,RNA was hierarchically clustered to determine the different m6 A methylation or expression patterns between samples.M6 A methylation quantitative detection kit was used to verify the change of m6 A expression level in cells after WTAP knockdown.The expression of CPSF4 and other downstream target genes was verified by RT-q PCR.In addition,the GEPIA2 tool was used to verify the correlation between downstream target genes and the prognosis of patients with esophageal squamous cell carcinoma,and the GSEA enrichment analysis tool was used to explore the main signal pathways of downstream target gene enrichment.Resluts:1.TCGA combined with GTEx database analysis results showed that m6 A methyltransferase WTAP and m6 A methyltransferase METTL3 m RNA expression levels were significantly up-regulated in esophageal cancer,and survival curve suggested that only WTAP was significantly correlated with poor prognosis of esophageal cancer patients.2.Immunohistochemical results showed that WTAP protein was highly expressed in esophageal squamous cell carcinoma compared with normal esophageal tissue.Statistical analysis showed whether the expression of WTAP was correlated with pathological stage and positive lymph node(P<0.001,P=0.008).Multivariate cox analysis showed that WTAP expression and pathological stage were independent prognostic factors(P=0.022,P=0.006).The WTAP knockdown plasmid was transfected into ESOPHAGEAL squamous cell carcinoma EC109 cells.The results showed that the expression level of m6 A,proliferation,migration and invasion ability of esophageal squamous cell carcinoma cells were significantly decreased after the expression of WTAP down-regulated,and the expressions of dry-related genes SOX2 and OCT4 were down-regulated(P < 0.05).3.In this study,WTAP gene was knocked down in esophageal cancer cells,and Arraystar epigenome chip was used for high-throughput sequencing.Sequencing results showed that m RNA expression of 612 genes was up-regulated and 2972 genes were down-regulated.Three up-regulated genes CPSF4,TAF3 and SMS related to the prognosis of esophageal cancer were screened by intersecting the sequencing results with the prognostic genes of esophageal cancer in GEPIA2.Among them,only CPSF4 m RNA expression was up-regulated and m6 A expression level was down-regulated(P<0.05).4.The results of this study showed that the overall m6 A level was down-regulated and CPSF4 expression was up-regulated after WTAP knockdown in esophageal cancer cells.In addition,CPSF4 m RNA level was upregulated after YTHDF2 was successfully knocked down in esophageal cancer cells(P<0.05),suggesting that YTHDF2 is a binding protein that WTAP regulates CPSF4 m6 A modification.The expression level of CPSF4 m RNA in esophageal cancer in TCGA database was used to enrich the GSEA pathway,and the P53 pathway was found to be in the forefront(DR<0.05,NES absolute value greater than 1,P<0.05).Therefore,it was speculated that CPSF4 may be highly enriched in the P53 pathway in esophageal cancer tissues.Conclusion: WTAP is overexpressed in esophageal squamous cell carcinoma and is associated with poor prognosis in patients with esophageal squamous cell carcinoma and promotes malignant phenotype of esophageal squamous cell carcinoma.WTAPmediated modification of CPSF4 m RNA m6 A reduces CPSF4 stability by relying on YTHDF2.The important role of WTAP in the m6 A methylation modification of esophageal carcinoma,and thus identified a new RNA regulatory mechanism.