Effects Of ATRX Knockdown On Proliferation And Of Glioma Stem Cells

Background:Malignant glioma is the most common intracranial malignant tumor with the highest mortality.At present,the treatment methods of malignant glioma mainly include surgical treatment,radiotherapy and chemotherapy which with poor treatment effect,the survival of most patients has not been significantly improved,especially the 5-year survival rate of Grade IV glioma patients with the highest degree of malignancy is less than 5%.Therefore,it is urgent to find a more effective treatment for glioma.Studies have shown that ATRX plays an important role in gene transcription,self-renewal,pluripotent maintenance and cancer development.ATRX is frequently mutated in GBM,but the role and molecular mechanism of ATRX in glioma remain unclear,so further research is needed.Objective:In this study,GSCs and P53-/-Pten-/-NSCs were selected as subjects.Then transfected GSCs and NSCs with sh ATRX.The effect of ATRX knockdown on the proliferation,differentiation,tumor growth and the survival of GSCs/NSCs orthotopic nude model by the vitro and vivo experiment,proving that ATRX is a tumor suppressor gene of malignant glioma.Method:In vivo and in vitro,the experiment groups were divided into the sh Rluc.713 group(negative control)and the sh ATRX group(sh ATRX.6144 and sh ATRX.7377).(1)Detection the expression of ATRX in GBM tissue microarray: tissue microarray analysis technology.(2)Western Blot assay was used to detect the effect of sh ATRX transfection on ATRX protein expression in GSCs.The effect of ATRX knockdown on the proliferation of GSCs: cell growth curve,soft agar colony formation assays,immunofluorescence staining.(3)The effect of ATRX knockdown on GSCs orthotopic nude model: establish GSCs orthotopic nude model.Immunohistochemical staining experiment was used to detect the effect of ATRX knockdown on the expression of ATRX,Ki67 and Nestin in tumor tissues of mice.(4)Western Blot assay was used to detect the effect of sh ATRX transfection on ATRX protein expression in NSCs.The effect of ATRX knockdown on the proliferation and differentiation of NSCs: immunofluorescence staining.Immunohistochemical staining experiment was used to detect the effect of ATRX knockdown on the differentiation and expression of Nestin in NSCs.(5)The effect of ATRX knockdown on NSCs orthotopic nude model: establish NSCs orthotopic nude model.Immunofluorescence staining was used to detect the effect of ATRX knockdown on the proliferation and differentiation of mouse tumor cells.Result:The low expression of ATRX protein in GBM samples was found by tissue microarray analysis.In vitro,knockdown of ATRX expression can promote the growth of GSCs and enhance the clone formation ability in differentiation condition.In vivo,ATRX knockdown promoted increased the expression of Ki67 and Nestin in tissues of GSCs orthotopic nude and shortened survival of mice.In vitro,knockdown of ATRX expression increased the expression of Brd U and Nestin of NSCs in differentiation condition.In vivo,ATRX knockdown shortened survival of orthotopic nude of NSCs.Conclusion:(1)ATRX knockdown can promote the proliferation,maintain the stemness of GSCs.In vivo,ATRX deletion can promote the growth of tumor and shortened survival of GSCs orthotopic nude model.(2)Knockdown of ATRX expression can promote the deterioration of P53-/-Pten-/-NSCs,then form tumors eventually.(3)Knockdown of ATRX expression promote GSCs proliferation by attenuating differentiation(4)ATRX is the tumor suppressor gene of malignant glioma.