METTL3 Regulates The Effect Of IFIT2 On The Proliferation And Invasion Of Esophageal Squamous Carcinoma Cells

Objective:This project takes the abnormal expression of METTL3 in clinical esophageal squamous carcinoma case samples as a starting point to investigate the association between METTL3 and clinical indicators such as esophageal squamous carcinoma development and metastasis,and to analyze and explore the possibility of m~6A RNA modification and METTL3 expression as a new molecular marker for esophageal squamous carcinoma.Methods:Clinical samples of esophageal squamous carcinoma from the Department of Thoracic Surgery of the First Affiliated Hospital of the University of Science and Technology of China were selected to study METTL3 protein expression in esophageal squamous carcinoma tissues and corresponding normal tissues adjacent to the carcinoma using immunohistochemistry(IHC),and then m RNA and protein expression levels were confirmed by q-PCR and Western Blot.Next,we identified the esophageal squamous carcinoma cell line with high METTL3 expression level as KYSE510 by Western Blot and transfected specific sh RNA to knock down METTL3 expression in KYSE510 cells.The effect of down-regulation of METTL3 on the proliferation of esophageal squamous carcinoma cells was analyzed using CCK8 and plate cloning assays.The effect of METTL3 on apoptosis of esophageal squamous carcinoma cells was analyzed by flow cytometry,and the effect of METTL3 on invasion and migration of esophageal squamous carcinoma cells was confirmed by scratch assay and invasion assay.A nude mouse model was used to study the regulation of METTL3 on esophageal squamous carcinoma growth in vivo:esophageal squamous carcinoma cell lines and control cell lines in which METTL3 was stably inhibited were constructed by transfecting with lentivirus-mediated METTL3-specific sh RNA and screened,and the same number of cells were injected subcutaneously in nude mice,and the change of tumor size in each group of mice was recorded every four days after injection.One month after injection,the tumors were weighed and photographed,and immunohistochemical staining was performed to confirm METLL3,Ki67(proliferating cell marker).The m~6A Me RIP-Seq technique was used to compare METTL3overexpressed KYSE510 cells and Scramble sequence transfected KYSE510 control cells,analyze their m~6A modification profiles,search for differentially modified genes,and identify IFIT2,a target gene specifically regulated by METTL3 expression for m~6A modification in esophageal squamous carcinoma.The biological functions of IFIT2 in esophageal squamous carcinoma cells were confirmed by CCK8,plate cloning,scratch assay and invasion assay.In addition,the si-IFIT2 vector was transfected in KYSE510cells to down-regulate IFIT2 expression levels and to test whether it could compensate for the phenotype after knockdown of METTL3 expression.The regulatory relationship between IFIT2 and METTL3 was confirmed functionally.Results:In clinical samples of esophageal squamous carcinoma,immunohistochemical assays showed that METTL3 was significantly highly expressed in paraneoplastic tissues,and q-PCR and Western Blot were consistent with the immunohistochemical results.At the cellular level,knockdown of METTL3 inhibited the proliferation,migration and invasion of esophageal squamous carcinoma cells and promoted apoptosis.At the animal level,knockdown of METTL3 inhibited the proliferative ability of subcutaneous tumors in mice.The results of high through sequencing showed that the downstream target gene of METTL3 was IFIT2.downregulation of IFIT2expression level could rescue the expression level of knockdown METLL3,while the proliferation and migration ability of esophageal squamous carcinoma cells were restored to some extent.Conclusion:We confirmed that the m~6A methylesterase METTL3 plays a pro-carcinogenic role in esophageal squamous carcinoma,and also found that METTL3mediates the downstream target gene IFIT2 to regulate esophageal squamous carcinogenesis and development,elucidating a new molecular mechanism of esophageal squamous cell carcinoma,which is of great value for early diagnosis and precise treatment of esophageal squamous carcinoma.