Repression Of Dok7 Expression Mediated By DNMT1 Promotes Glioma Cells Proliferation

Background:Glioma is one of the most common primary malignant tumors in the central nervous system.Its high incidence,easy recurrence after surgery,low 5-year survival rate,and poor prognosis are its main features.The current treatment of glioma is mainly surgical treatment combined with postoperative radiotherapy and chemotherapy(for example: temozolomide),but the treatment effect is not satisfactory.Therefore,in-depth study of the molecular mechanism of the occurrence and development of glioma can provide new ideas and methods for the treatment of glioma,but the molecular mechanism of glioma occurrence and development is still largely unclear.Previous studies have shown that the expression of Dok7 in human glioma tissues is significantly down-regulated relative to the expression in normal brain tissue,and Dok7 overexpression significantly inhibits proliferation and colony formation of glioma cell lines in vitro.This provides a new direction for our next research.Based on the above findings,we cannot help but ask: First,since Dok7 overexpression inhibits the proliferation of glioma cells in vitro,whether Dok7 overexpression also inhibits the proliferation of glioma cells in vivo;Second,Dok7 What is the expression of human glioma tissue that is lower than that of normal brain tissue?Methods:1.The glioma cell lines U87 and U251 were cultured in vitro,and Dok7 overexpressing(Dok7)and vector stable cell lines were established in U87 and U251 cell lines to study the proliferative ability of Dok7 overexpressing cell line U251 in vitro.2.In the nude mice,the tumors were subcutaneously inoculated with Dok7 overexpressed or empty U87 stably transfected cells in the left axilla of female nude mice,and the tumor growth of each group was recorded in detail.The volume of nude mice tumors was statistically analyzed.After 40 days,different groups of tumors were taken for immunohistochemical analysis to study the effect of Dok7 overexpression on glioma proliferation in vivo.3.In vitro cultured glioma cell lines U87 and U251 were respectively added with DNMT1 inhibitor 5-Aza to study the effect of 5-Aza on glioma cell methylation and expression of Dok7 m RNA and protein levels influences.4.The DNMT1 knockdown plasmid was transfected into the glioma cell lines U87 and U251 cultured in vitro to study the effect of DNMT1 on methylation of glioma cells and the m RNA and protein levels of Dok7.Result:1.Overexpression of Dok7 inhibits the proliferation of glioma cell line U251 in vitro.2.The weight and volume of tumors in nude mice of Dok7 overexpression group were significantly lower than those in empty group;3.The methylation of U87 and U251 cells with DNMT1 inhibitor 5-Aza was significantly lower than that of U87 and U251 cells without 5-Aza.The expression of Dok7 m RNA and protein were significantly higher in 5-Aza group than in control group;4.After transfected with DNMT1 knockdown plasmid,the methylation level of glioma in sh-DNMT1 group was significantly lower than that in sh-con group.The expression of Dok7 m RNA and protein in sh-DNMT1 group was significantly higher than in Shcon group.Conclusion:1.Dok7 inhibits the proliferation of glioma cells in vitro and in vivo;2.DNMT1 mediates the methylation of Dok7 and inhibits the expression of Dok7.