HAX-1 Regulates The Proliferation And Apoptosis Of Glioma Cells Through Akt Pathway Modulation

Background and Objective:Glioma is the most common malignant tumor of the central nervous system,and it is still one of the difficulties in the treatment of nervous system diseases.Especially for patients with high-grade gliomas with high nausea,the survival rate of patients is still at a low level.Therefore,it is particularly urgent to explore the mechanism of glioma incidence and prognosis in order to explore new therapeutic modalities for glioma.Hematopoietic stem cell specific protein 1-related protein X-1(HAX-1)is an anti apoptotic protein discovered in recent years,which is over expressed in many tumor cells,and promotes tumor cell proliferation,migration and inhibition of tumor cell apoptosis.HAX-1 protein has many biological functions,but its research in glioma is still blank.In this study,we will take HAX-1 as the breakthrough point to study the role of HAX-1 in glioma proliferation and apoptosis,and preliminarily explore the mechanism of HAX-1.Methods:1.cell culture:cultured human glioma cells U118,U87-MG,U251 and SHG44,and human glial cells HA.2.immunohistochemical staining:the expression of HAX-1 in the tissue of human glioma was determined.3.qPCR Technology:To detect the transcriptional level of HAX-1 in glial cells and glioma cells,and the level of HAX-1 transcription in glioma tissue and paracancerous tissue,and the transcription level of p53 protein in glioma cells U118 and U87-MG.4.Western blot:to detect expression of HAX-1 in human glioma tissues and paracancerous tissue;the expression of HAX-1 in glioma cells and glial cells;detection in the expression of HAX-1 in HAX-1 knocked out U118 and U87-MG;the expression of p21,BAX,p53 and Hsp90 protein in glioma cells U118 and U87-MG;the activation of Caspase9,Caspase3 and PARP protein in glioma cells U118 and U87-MG under oxidative stress conditions;the degradation and ubiquitination level of p53 protein in glioma cells U118 and U87-MG;the changes of the phosphorylation level of Aktl protein and MDM-2 protein in U118 and U87-MG glioma cells;the changes of interaction of Aktl and Hsp90 in glioma cells U118 and U87-MG.5.the clone formation test was used to detect the changes of the clone formation ability before and after the HAX-1 knockout.6.Edu cell proliferation test:using Edu to mark proliferating cells to evaluate the proliferation of glioma cells before and after HAX-1 knockout.7.flow cytometry:the changes in the cell cycle of glioma cells U118 and U87-MG before and after HAX-1 knockout were detected by flow cytometry.8.flow cytometry:the changes in the apoptosis of glioma cells U118 and U87-MG before and after HAX-1 knockout were detected by flow cytometry.9.Co-immunoprecipitation:Co-immunoprecipitation was used to detect the binding of HAX-1 to Hsp90 protein in glioma cells U118 and U87-MG.The ubiquitination level of p53 protein in U118 and U87-MG cells before and after HAX-1 knockout,and the interaction between Aktl and Hsp90 protein were also changed.10.immunofluorescence co-localization staining:immunofluorescence co-localization staining was used to detect the distribution and co-localization of Hsp90,HAX-1 and Aktl proteins in U118 and U87-MG cells.Results:Through statistical analysis of a series of experiments and related results,we found that HAX-1 protein showed higher expression in glioma tissues and cells,and the expression of HAX-1 is related to with tumor size,differentiation degree and pathological grade of glioma and is the independent prognostic factors of glioma.We found that HAX-1 protein knockout can inhibit glioma cells U118 and U87-MG clone forming ability after regulating of the expression of HAX-1 in U118 and U87-MG cells,and the HAX-1 knocked-out also reduce the proportion of proliferating cells and leads U118 cells and U87-MG cells arrest in G0/G1 phase,and also leads to increased expression of p21.HAX-1 protein knockout also increased the apoptosis rate of U118 and U87-MG in H202 simulated oxidative stress environment,and increased the activation level of apoptotic protein such as Caspase9,Caspase3 and PARP.Accordingly,the expression of apoptosis related proteins,such as p53 protein and downstream protein BAX,was increased after HAX-1 knockout.Further analysis showed that HAX-1 protein did not affect the transcription of p53 protein in U118 and U87-MG,but promoted the degradation of p53 protein by affecting the ubiquitination of p53 protein.At the same time in U118 and U87-MG cells,HAX-1 protein decreased the phosphorylation of p53 protein ubiquitin E3 ligase MDM-2 and the MDM-2 upstream protein Aktl,and the overexpression of HAX-1 enhanced the phosphorylation level of MDM-2 and Aktl correspondingly.Then we verified that HAX-1 can bind to Hsp90 in glioma cells U118 and U87-MG,and find that HAX-1 can promote the binding of Hsp90 to Aktl protein by binding with Hsp90 protein,which promotes the level of phosphorylation of Aktl protein.The effect of HAX-1 protein on the phosphorylation level of Aktl and MDM-2 protein was weakened after the use of Hsp90 protein selective inhibitors.Conclusion:In conclusion,after our research,we found that HAX-1 protein enhanced glioma cell proliferation and anti apoptosis ability.