Studies On The Mechanism Of Radioresistance Regulated By C1GALT1 In Esophageal Cancer

Background:Esophageal cancer is a common malignant tumor of digestive system,and the morbidity and mortality of esophageal cancer in China are among the highest in the world.The onset of esophageal cancer is insidious.More than 90%of patients have advanced to mid-advanced stage at the time of diagnosis.The quality of life is poor and the 5-year survival rate is only 29.7%.At present,the treatment of early,middle and advanced esophageal cancer is mainly surgery or radiotherapy,and advanced esophageal cancer is often treated with combined chemotherapy and radiotherapy,so radiotherapy plays an important role in the treatment of esophageal cancer.However,the root cause of radiotherapy failure is radiotherapy resistance.Due to radiotherapy resistance,the local esophageal cancer is not controlled or the recurrence rate reaches about 80%after radiotherapy.Therefore,elucidating the molecular mechanism of radioresistance in esophageal cancer is the key to improving the efficacy of radiotherapy and improving the prognosis of patients.Our group found that the overexpression of C1GalT1 was related to the occurrence of radioresistance in laryngeal cancer cells.Immunohistochemical(IHC)results and data from public database indicate that C1GalT1 is more overexpressed in esophageal cancer tissues than that in tumor-adjacent normal tissues.Therefore,to clarify C1GalT1 and its functions is another breakthrough to solve the radiation resistance of esophageal cancerObjective:To investigate the radioresistance of C1GalT1 on human esophageal carcinoma cell line and its mechanismMethods:Immunohistochemistry was used to analyze the correlation between the expression of C1GalT1 and pathological characteristics and prognosis of patients The expression of C1GalT1 after transfection with C1GalT1 SiRNA was analyzed by qPCR and western blot.Then flow cytometry was used to detect the apoptosis and cell cycle distribution after C1GalT1 SiRNA.Clone formation technique was used to detect the effect of C1GalT1 SiRNA on cell proliferation.The effect of radiation with low dose on cell invision and migration ability was analyzed by Transwell technique and scratch technique.Flow cytometry and lectin technology were applied to detect the expression of core type 1 oxyglycan after transfection of C1Ga1T1 SiRNA,and lectin pull-down technology were used to analyze the binding of β 1-integrin and Jacalin Immunoblot assay was applied to detect the expression of β 1-integrin after transfection with C1GalT1 SiRNA.Western Blot test was used to detect p-FAK expression after transfection with C1GalT1 SiRNA,β1-integrin,p-FAK and p-Akt expression after P4C10 pretreatment and the expression of p-FAK and p-Akt after pretreatment with FAK inhibitors.The effect of radiation on apoptosis after treatment with P4C10 or FAK inhibitors was detected by floe cytometryResults:Immunohistochemistry results showed that C1GalT1 was significantly higher expressed in esophageal cancer than tumor-adjacent normal tissues.High expression of C1GalT1 was correlated with TNM staging(P<0.05),lymph node metastasis(P<0.05)and poor prognosis.There was no significant correlation between C1GalT1 expression and gender,age,tumor size,invasion depth and differentiation QPCR and Western Blot indicated that the expression of C1GalT1 decreased in the three kinds of C1GalT1 SiRNA transfection,among which SiRNA-3 had the best transfection effect.Flow cytometry experiments confirmed that after interfering with C1GalT1 SiRNA,radiation promoted cell arrest in G0/G1 phase and reduced cell arrest in G2/M phase.After interfering with C1GalT1,radiation promoted cell apoptosis.The results of clone formation experiments suggested that cell proliferation was inhibited when interfered with C1GalT1 at various dose point.Transwell technology and scratch experiments suggested that interference with C1GalT1 inhibited low dose radiation-induced cell invision and metastasis.Flow cytometry and lectin experiments confirmed that the expression of core type 1 oxyglycan decreased after downregulation of C1GalT1.Lectin pull down and immunoblotting confirmed that after transfection with C1GalT1 SiRNA,the binding of β 1-integrin to Jacalin decreased,but the expression of β 1-integrin did not change significantly.Western blot experiments confirmed that the expression of p-FAK decreased after transfection with C1GalT1 SiRNA.After P4C10 pretreated cells,the expression of β1-integrin,p-FAK and p-Akt all decreased.After cell treated with FAK inhibitors,the expression of p-FAK and p-Akt were reduced,and the results of flow cytometry confirmed that the radiosensitivity of cell was enhanced after treatment with P4C10 or FAK inhibitorsConclusion:In summary,the overexpression of C1GalT1 inhibited the radiosensitivity of the esophageal cancer cells,and the intervention of the expression level of C1GalT1 could affect the radiosensitivity of the esophageal cancer cells C1GalT1 could activate the FAK pathway by increasing the level of 1-integrin,thus participating in the radiosensitivity of esophageal cancer radiotherapy.The experiment laid the foundation for studying the mechanism of C1GalT1’s involvement in radiotherapy sensitivity,and provided potential drug targets for improving the clinical radiotherapy effect.