The Effect And Mechanism Of ATP1A1 On Proliferation And Apoptosis In Human Glioma Stem Cells

BACKGROUND: Glioblastoma(GBM)is the most common primary malignant brain tumor with rapid proliferation,invasiveness,poor therapeutic effect,high recurrence rate and short survival time.At present,basic research on brain tumors has made great progress,but these advances have not significantly improved the prognosis of patients.In the case of traditional therapeutic intervention,the average overall survival of GBM patients is only about 15 months.Glioma stem cells(GSCs)have been extensively studied since their discovery.Although GSCs are only a small subset of gliomas,they are significantly different from common tumor cells because of their self-renewal and immortalization.Multipotential differentiation and the potential to rebuild tumors are the key to maintaining and promoting tumor growth,and in a certain sense determine the chemoresistance and tumor recurrence of tumors.Therefore,targeting GSCs may provide a more effective treatment for patients with high-grade gliomas.In order to understand the specific molecular mechanism of glioma stem cells differentiated from differentiated common glioma cells,we compared the glioma stem cells relative to their differentiated cells by proteomics using iTRAQ combined with 2D LC-MS/MS.Specific differential proteins,found that ATP1A1 is abnormally highly expressed in GSCs,may be a potential target for targeting glioma stem cells.METHODS: In this study,in order to further understand the role of ATP1A1 in the malignant phenotype and pathogenesis of primary human GSCs,we first isolated and cultured primary human glioblastoma stem cells and cultured them.The expression of ATP1A1 in different grades of gliomas was detected,and the expression of ATP1A1 in GSCs and differentiated GBM cells was detected.Finally,two primary GSCs were selected for subsequent studies.Subsequently,ATP1A1 lentivirus-interfering GSCs were constructed,and the proliferation and apoptosis of the interference group(sh-ATP1A1 GBM GSCs)and the control group(sh-NC GBM GSCs)and the ability to form tumors in vivo were detected.Detection of sh-ATP1A1 GBM GSCs /sh-NC changes in MAPK and PI3 K signaling pathways in GBM GSCs and detection of ATP1A1 acting on the important node Src of MAPK and PI3 K signaling pathway;detecting the interaction between ATP1A1 and Src and the change of Src phosphorylation level after interfering with ATP1A1 expression;Using sh-Src lentivirus,the expression of Src was interfered in GSCs that restored ATP1A1 expression,and the proliferation and apoptosis of cells after Src down-regulation were detected.RESULTS: We successfully isolated glioma stem cells from human primary glioblastoma tissue,and allowed GSCs to adhere to the monolayer by pre-coating of fibronectin and polylysine.Sub-spherication ability and expression of stem cell markers SOX2 and NESTIN;cells differentiated and expressed differentiation marker GFAP.In addition,we found that ATP1A1 is more preferentially expressed in high-grade gliomas,and ATP1A1 expression is significantly up-regulated in stem cells relative to glioma cells;knockdown of ATP1A1,GSCs proliferation is inhibited,cell cycle arrest,apoptosis increased In vivo,tumorigenic ability decreased;p-AKT and p-ERK1/2 activity decreased;Src is a key node of ATP1A1 regulating AKT and ERK;and ATP1A1 has direct or indirect interaction with Src,and Src activation in GSCs after ATP1A1 down-regulation The level was also down-regulated;after the expression of Src was down-regulated in the GSCs that restored ATP1A1 expression,the cells showed inhibition of proliferation and increased apoptosis.Conclusion: ATP1A1 is highly expressed in GSCs and can regulate the proliferation and apoptosis of GSCs.It may affect the Src-mediated MAPK and PI3 K signaling pathway activities by directly or indirectly interacting with Src to affect the phosphorylation of Src.Proliferation and apoptosis.