The Study On The Biological Function Of FTO In The Development Of Ewing's Sarcoma

Objective & Background:Ewing's sarcoma is a highly invasive malignant tumor,which has a high incidence in adolescents.Due to its early metastasis,easy recurrence and poor prognosis,Ewing's sarcoma is seriously harmful to adolescent health.Many studies have shown that,FTO(Fat Mass and Obesity Associated)gene is closely related to m6 A methylation and has been expressed in breast cancer,gastric cancer,acute myeloid leukemia(AML)and other tumors.And involved in the occurrence,development,invasion and metastasis of a variety of tumors,is an important focus of targeted therapy research in the future.However,the expression of FTO in Ewing's sarcoma and its relationship with the malignant biological behavior of Ewing's sarcoma have not been reported in the literature.The role of phosphatase and tensin homolog(PTEN)as a typical tumor suppressor in Ewing's sarcoma has rarely been reported.The purpose of this study was to investigate the expression of FTO gene in Ewing's sarcoma and its effect on the biological function of Ewing's sarcoma cells,and whether the tumor suppressor gene PTEN in the PI3K/AKT signaling pathway was involved in the carcinogenesis of FTO in Ewing sarcoma,to explore its role in the occurrence and development of Ewing's sarcoma,to provide experimental basis for finding suitable target for targeted therapy of Ewing's sarcoma.Methods:Realtime-qPCR was used to detect the expression of FTO and PTEN in Ewing's sarcoma tumor tissue and paratumor tissue,as well as in Ewing's sarcoma cell lines and normal osteoblasts.A stable FTO overexpression or knockout Ewing's sarcoma cell lines were constructed,and the stability of the cell lines were verified by RT-qPCR and Western blot.CCK-8 assay,scratch test and Transwell assay were used to detect the changes of proliferation,migration and invasion of Ewing sarcoma cell line after FTO overexpression or knockout,respectively.The expression of PTEN of Ewing sarcoma cell line after FTO overexpression and knockout were detected by Realtime-qPCR and Western blot.The methylated m6 A RNA immunoprecipitation(me-RIP)was performed to evaluate the level of mRNA methylation modification of PTEN in Ewing sarcoma cellline after FTO overexpression and knockout.The stability of PTEN mRNA was analyzed by transcriptional inhibition test.CCK-8 assay was used to detect the effect of PTEN overexpression on the proliferation of Ewing sarcoma cell line with FTO overexpression.Results:The expression of FTO in Ewing's sarcoma tumor tissue and Ewing's sarcoma cell line was significantly higher than that in paratumor tissue and normal osteoblasts,while the expression of PTEN was on the contrary.There was a negative correlation between FTO and PTEN.The proliferation at 72 h,relative migration distance and the number of invasive cells of Ewing's sarcoma cells overexpressed with FTO were significantly higher than those of Ewing's sarcoma cell line(control group),but the expression levels of mRNA and protein of PTEN,the mRNA methylation modification and the stability of mRNA were significantly lower.While in Ewing's sarcoma cell line with FTO knockout,the outcome was on the contrary.After the recovery of PTEN,the promoting effect of FTO on the proliferation of Ewing sarcoma cells was reversed.The difference was statistically significant.Conclusion:The up-regulation of FTO expression in Ewing's sarcoma is related to the occurrence and development of Ewing's sarcoma.Up-regulation of FTO expression can promote the proliferation,migration and invasion of Ewing's sarcoma cells,while down-regulation of FTO expression results in the opposite.FTO may promote the proliferation of Ewing sarcoma by depressing the mRNA methylation of PTEN.