Expression Of The M~6A Binding Protein YTHDF1 In Esophageal Squamous Cell Carcinoma And Its Relationship With Radiosensitivity

Objective:The morbidity and mortality of esophageal squamous cell carcinoma(ESCC)are high in China,and most of the patients are in the middle and advanced stage.Radiotherapy is one of the important treatments for advanced esophageal cancer.Radio-resistance is an important factor affecting the prognosis of patients.Therefore,there is an urgent need to find molecular markers that can regulate and improve the radiosensitivity of ESCC.m6A is the most abundant methylation modification in eukaryotic cells.The purpose of this study is to explore the relationship between YTHDF1 and radiosensitivity of ESCC.By knocking down the expression level of YTHDF1 in esophageal squamous cell carcinoma cells and then irradiation to observe the effect on the function of esophageal squamous cell carcinoma cells,to provide experimental evidence for its effect on radiosensitivity of ESCC in vitro,to establish the relationship between m6A and radiotherapy of ESCC,and to explore its mechanism.Methods:By analyzing TCGA and GTEx database,screening the expression of YTHDF1 in ESCC,analyzing cBioPortal database,screening ESCC patients undergoing radiotherapy and analyzing the effect of YTHDF1 on survival time.Specimens of ESCC and adjacent tissues were collected,and the expression of YTHDF1 protein was detected by immunohistochemical experiment.The expression of YTHDF1 was knocked down by cell transfection,and the knockdown efficiency of YTHDF1 was detected by RT-qPCR and Western blot.The colony number of esophageal cancer cells was counted by colony formation test,and the survival curve was constructed by linear quadratic model.The survival fraction of esophageal squamous cell carcinoma cells at different irradiation doses was calculated to detect the effect of YTHDF1 on the colony formation ability of esophageal cancer cells after irradiation.Esophageal squamous cell carcinoma cell lines were divided into control,siYTHDF1,radiotherapy and siYTHDF1+radiotherapy groups,the proliferation of cells in different groups was detected by CCK-8 and EdU assays.The apoptosis of cells was detected by AnnexinV/PI double staining method combined with flow cytometry,and the expression of y-H2AX was detected by cellular immunofluorescence staining to analyze the damage of DNA double-strand breaks.The downstream binding sites of YTHDF1 were screened by m6A sequencing,KEGG and GO analysis and YTHDF1-RIP-qPCR,and the expression of downstream targets in esophageal squamous cell carcinoma was analyzed by immunohistochemistry.Results:A query in the TCGA+GTEx database revealed that YTHDF1 was highly expressed in esophageal squamous cancer tissues(P<0.05),and futher analysis of the cBioPortal database revealed that the higher YTHDF1 expression in patients with ESCC treated with radiotherapy was associated with shorter survival(P<0.01).The median immunohistochemical score of YTHDF1 in 25 esophageal squamous cell carcinoma tissues was 6,and the high expression rate was 72.0%,which was higher than that of the paraneoplastic tissues(P<0.05).After transfecting esophageal squamous carcinoma cell lines with small interfering RNA(siRNA),YTHDF1 was significantly lower in mRNA and protein levels compared with the control group,respectively(P<0.05).YTHDF1 knockdown esophageal squamous carcinoma cell lines were given irradiation doses of 0Gy,2Gy,4Gy,6Gy,and 8Gy,and the decrease in cell survival fraction was found to be more pronounced with increasing irradiation dose by clone formation assay.Knockdown of YTHDF1 inhibited the proliferation of esophageal cancer cells(P<0.05),and the proliferation of combined 6Gy irradiated esophageal cancer cells was further decreased(P<0.01).The results of Annexin V/PI double staining method suggested that the effect of YTHDF1 knockdown on apoptosis was not statistically significant(P>0.05),and the knockdown of YTHDF1 combined with 6Gy irradiation treatment significantly increased apoptosis compared with the control group(P<0.01),and the cell immunofluorescence analysis revealed that the y-H2AX protein formation was significantly increased after 4h and 24h of YTHDF1 knockdown compared with the control group,demonstrating an increase in apoptosis.Bioinformatics analysis,m6A sequencing,KEGG and GO analysis,and YTHDF1-RIP-qPCR validation indicated that AURKA was positively correlated with YTHDF1 and immunohistochemical results suggested that AURKA protein was highly expressed in esophageal cancer tissues.Conclusion:The high expression of YTHDF1 in ESCC tissues was consistent with the results analyzed in the database.YTHDF1 knockdown affects the colony formation,cell proliferation and apoptosis of ESCC cells after irradiation.YTHDF1 may be involved in affecting the radiosensitivity of esophageal squamous cell carcinoma cells,and may bind to AURKA.YTHDF1 may be a new target to improve the sensitivity of esophageal radiotherapy.