| Background:According to the latest cancer statistics report,esophageal cancer(EC)is the seventh most common malignancy with the sixth-highest overall mortality rate.Esophageal cancer includes esophageal squamous cell carcinoma(ESCC),which accounts for about 95 percent of all esophageal cancers,as well as esophageal adenocarcinoma.Despite considerable progress in cancer diagnosis and treatment,the majority of patients with advanced ESCC still show a poor prognosis due to the high rate of recurrence and metastasis,with an overall 5-year survival rate of less than 15%for ESCC.Therefore,it is of great significance to elucidate the underlying biological mechanism of ESCC for clinical diagnosis and the development of effective treatment strategies.N6-methyladenosine(m6A)modification is one of the most abundant RNA modifications,and m6A modification is present in about 25%of mRNAs in mammals.m6A modification mainly regulates the stability,splicing,transport,and translation of mRNA at the post-transcriptional level.m6A methyltransferase is responsible for the formation of RNA m6A modification,of which the most important catalytic subunit is methyltransferase-like 3(METTL3).Recent studies have reported that METTL3 acts as an oncogene in several types of cancer,including colorectal cancer,ovarian cancer,lung adenocarcinoma,melanoma,and cervical cancer.METTL3 plays a key role in the development and biological process of cancer,including cell proliferation,apoptosis,invasion,migration,etc.However,the regulatory mechanism of METTL3 in the occurrence and development of ESCC remains to be determined.Metabolic reprogramming is considered an emerging marker of cancer cells.Warburg effect indicates that cancer cells still prefer glycolysis metabolism to meet the energy and biosynthesis requirements of continuous proliferation even in an oxygen-rich environment.Activation of proto-oncogenes or inactivation of tumor suppressor genes can regulate the glycolysis process and affect tumor occurrence and progression,METTL3 has been reported to regulate glycolysis metabolism in colorectal cancer,gastric cancer and other tumors.So,is METTL3,which acts as a carcinogen in most tumors,involved in the regulation of the ESCC glycolysis pathway?Therefore,this study aims to investigate the effect of METTL3 on glycolysis of ESCC cells and its possible molecular mechanism,to provide laboratory basis for the exploration of new clinical therapeutic targets.Objective:1.To analyze the expression level of methyltransferase METTL3 in ESCC tissues and cells,and its potential impact on ESCC biological functions;2.To investigate the effect of METTL3 on the glycolysis pathway in ESCC cells and its potential molecular mechanism.Methods:1.The expression of METTL3 in different types of cancer,especially esophageal cancer,was analyzed based on TCGA database.The diagnostic value of METTL3 for ESCC was predicted by drawing the working curve of subjects.2.Immunohistochemistry was used to detect the expression of METTL3 in ESCC tissues and adjacent tissues.Western Blot was used to detect the expression of METTL3 in ESCC cells.3.METTL3 silenced and overexpressed vectors were constructed and transfected into ESCC cell lines KYSE150 and TE1 to construct stably silenced METTL3 and stably overexpressed METTL3 cell lines.4.Through the CCK-8 and colony formation experiments testing the proliferation ability in METTL3 silenced and overexpressed KYSE150/TE1 cells.5.Through the wound healing and Transwell experiments testing the cell migration ability in METTL3 silenced and overexpressed KYSE150/TE1 cells.6.Through Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes,KEGG enrichment analysis and Gene set Enrichment analysis(GSEA)were performed to analyze the possible biological and molecular pathways of METTL3 regulating ESCC cell malignant proliferation.7.Changes in Glucose and Lactate in METTL3 silenced and overexpressed KYSE150/TE1 cells using Detection Kits.8.Real-time PCR and Western Blot experiments were used to analyze the expression levels of GLUT4 and LDHA in the METTL3 silenced and overexpressed KYSE150/TE1 cells.Western Blot was used to analyze the effects of METTL3 on the levels of related proteins in mTORC1 signaling pathway.9.The expression of GLUT4 was reduced in METTL3 silenced KYSE150/TE1 cells by small interfering RNA.The effect of GLUT4 reduction on ESCC cells was verified by CCK-8 assay,and the effect of GLUT4 silenced on mTORC 1 signaling pathway-related protein levels was verified by Western Blot.10.The effects of lactate on METTL3 silenced KYSE150/TE1 cell proliferation were detected by colony formation and CCK-8 assay,and the effects of lactate on the levels of mTORC 1 signaling pathway-related proteins were verified by Western Blot.11.The effects of Rapamycin on METTL3-silenced KYSE150/TE1 cell proliferation were detected by colony formation and CCK-8 assay,and the effects of Rapamycin on mTORC1 signaling pathway--related protein levels were verified by Western Blot.Results:1.TIMER database analysis showed that the mRNA level of METTL3 was up-regulated in various types of cancer tissues compared with normal tissues,and the mRNA level of METTL3 was significantly increased in esophageal cancer tissues(P<0.001).2.Analysis of METTL3 transcriptional level in the TCGA-based database showed that METTL3 was significantly overexpressed in ESCC,and the 5-year survival rate predicted by the ROC curve showed that METTL3 had a high diagnostic efficiency for ESCC.3.Immunohistochemical results showed that METTL3 was highly expressed in ESCC clinical cancer tissues compared with para-cancer tissues,and Western Blot results showed that METTL3 was highly expressed in ESCC cell lines.4.Real-time PCR and Western Blot results showed that METTL3 silenced could effectively inhibit METTL3 mRNA(P<0.05)and protein expression in KYSE150/TE1 cells.Overexpressed of METTL3 significantly increased METTL3 mRNA(P<0.05)and protein expression in KYSE150/TE1 cells.5.The results of CCK-8 and colony formation experiments showed that METTL3 silenced could significantly inhibit the proliferation of KYSE150/TE1 cells;On the contrary,overexpressed of METTL3 could significantly promote the proliferation of KYSE150/TE1 cells.6.Wound healing and Transwell results showed that METTL3 silenced could significantly inhibit the migration of KYSE150/TE1 cells;On the contrary,overexpressed of METTL3 significantly promoted the migration of KYSE150/TE1 cells.7.GO and KEGG pathway enrichment analysis of METTL3 showed that METTL3 in ESCC may promote the proliferation of ESCC cells through metabolism and other pathways;Further GSEA pathway enrichment analysis of METTL3 showed that METTL3 was significantly positively correlated with glycolysis metabolism and mTORC1 pathway.8.Compared with the control group,METTL3 silenced cell lines reduced glucose consumption and lactate release;Overexpressed of METTL3 cell lines increased glucose consumption and lactate release.9.Real-time PCR and Western Blot results showed that GLUT4 and LDHA expression levels were decreased in METTL3 silenced ESCC cell lines,overexpressed of METTL3 significantly promoted the expression levels of GLUT4 and LDHA.Western Blot results showed that METTL3 silenced significantly inhibited the expression of related proteins in the mTORC1 signaling pathway,overexpressed of METTL3 significantly promoted the expression of mTORC1 signaling pathway-related proteins.10.Western Blot results showed that small interfering RNA successfully reduced the expression of GLUT4 in METTL3 silenced KYSE150/TE1 cells,and significantly inhibited the expression of mTORC1 signaling pathway related proteins.The detection of metabolites and CCK-8 assays showed that the ability of material metabolism and proliferation was also significantly reduced11.Colony formation assay and CCK-8 assay showed that lactate supplementation rescued the proliferation of METTL3 silenced ESCC cells;Western Blot results showed that lactate supplementation rescued the expression of mTORC1 signaling pathway-related proteins in silencing METTL3 ESCC cells.12.Colony formation assay and CCK-8 assay showed that Rapamycin more significantly inhibited the proliferation of METTL3 silenced ESCC cells;Western Blot results showed that Rapamycin more significantly inhibited the expression of mTORC1 signaling pathway-related proteins in METTL3 silenced ESCC cells.Conclusion:1.METTL3 was highly expressed in ESCC,and METTL3 promoted the proliferation and metastasis of ESCC cells;2.METTL3 is involved in regulating glycolysis in ESCC;3.METTL3 regulates the expression of downstream target GLUT4 and promotes the malignant proliferation of ESCC cells by activating the mTORC1 pathway.METTL3 is a potential therapeutic target for ESCC. |
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