| Glioma is the most common primary malignancy of the central nervous system,accounting for 27% of all central nervous system tumors,or 80% of intracranial malignant tumors.It is one of the most intractable tumors in human beings.Although surgery combined with chemoradiotherapy treatment strategies have made progress in recent years,the latest Central Brain Tumor Registry of the United States(CBTRUS)showed the five-year survival rate of glioblastoma is still only 5.5%.The complex heterogeneity and inhomogeneity within or among tumors are the main cause of its resistance to various physical and chemical factors and the recurrence of the tumor.Glioma stem cells(GSCs)is a subgroup of tumor cells with self-renewal,unlimited proliferation and multi-differentiation properties,GSCs usually expressed certain characteristics of stem cell phenotypic molecular,and often distributed in the periphery of tumor necrosis area,microvasculature,and the tumor edge,is considered to be the protagonist of tumor progression and recurrence.Therefore,the biological behavior of glioma stem cells,such as self-renewal,invasion and migration,and its role in the progression and recurrence of glioma have become the hot spots of current research.Recent studies found that Notch1 signaling is highly activated in glioma stem cells,Notch1 signaling pathway was found to play an important role in the regulation of glioma stem cell proliferation,differentiation,self-renewal behavior.Different from the cell signaling pathway activated by the binding of the dissociative ligand to the cell surface receptor,the ligands and receptors of the Notch pathway are all transmembrane proteins on the cell surface.Only when the receptor contacts with the neighboring ligand cells,the combination of ligand and receptor can be realized to start the Notch signaling.Through the hydrolysis of gamma secretase,the Notch intracellular domain(Notch intracellular domain,NICD)was released and entered the nucleus to interact with the transcription co-regulating factor CBF1,activating downstream target genes Hes and Hey,such regulating the cell outcome.The Notch pathway is abnormally activated in human brain cancers,Notch1 could maintain the stem phenotype,inhibit the differentiation and promote the proliferation of glioma stem cells.It has been found that activation of Notch pathway can initiate Epithelial-mesenchymal transition(EMT),thereby enhancing migration and invasion ability in pancreatic cancer,esophageal cancer,breast cancer and other tumors.Chemokine receptor 4(C-X-C chemokine receptor type 4,CXCR4)was originally known as a co-receptor in purified HIV.Recently,it has been found that CXCR4 is expressed in many tumor cells.In glioma,CXCR4 is also highly activated and correlated with tumor grades and poor patient prognosis.Compared to patients with low expression of CXCR4,a higher expression of CXCR4 in glioma patients may have a worse prognosis after radiotherapy and chemotherapy.CXCR4 can specifically bind to its ligand SDF-1,also known as stromal cell derived factor(CXCL12),and plays an important role in chemotaxis,migration and angiogenesis of malignant glioma cells.Compared with primary cultured glioma cells,CXCR4 was found enriched in GSCs.Specifically,on the one hand,CXCR4 could promote the formation of invadopodia and matrix metalloproteinase secretion to contribute to the invasion and migration of glioma stem cells.On the other hand,CXCR4 could promote the encoding transcription of proliferation,migration and invasion of GSCs through PI3k/ AKT and ERK pathways.In addition,the autocrine or paracrine of CXCL12 could also maintain the stem phenotype and the proliferation state of glioma stem cells.Our previous study found that Notch1 signaling molecular and CXCR4 were coexpressed in glioblastoma tissues.Both of them were corelated with pathological grade at the protein level,mRNA expression profiles for glioma cell lines(including glioma cell lines and glioma initiating cell lines)also showed that Notch1 and CXCR4 were enriched in glioma stem cells compared with traditional glioma cell lines,suggesting that there might be a crosstalk between Notch1 and CXCR4 to regulate the biological behavior of glioma stem cells.The aim of this study is to investigate the role and potential regulation mechanism of Notch1 signaling pathway in the self-renewal and invasion of glioma initiating cells.This research included following four parts.1.The expression level of Notch1 signaling molecular in glioblastoma and its association with patients’ prognosis.GEPIA online database showed that Notch1 is highly expressed in GBM compared to other tumor tissues.Bioinformatics was used to retrieve Oncomine database platform Bredel Brain 2 and Sun Brain mRNA data and showed that Notch1 expression is highly expressed in glioblastoma tissues compared with control brain tissues.TCGA mRNA and clinical data was retrieved to performed Kaplan-Meier survival curve,the results showed that high Hes1 expression correlated with a poor patient survival.GEO mRNA expression profiles of Notch1 and CXCR4 in glioma stem cell lines and conventional glioma cell lines revealed that Notch1 and CXCR4 are enriched in GICs and shared a co-expression trend.2.The effect of Notch1 on the invasion,migration and self-renewal of glioma initiating cells.We used lentivirus transfection to establish a glioma cell line with stably knockdown expression of Notch1 protein.The glioma cell spheres were obtained through stem cell culture medium,and CD133+ glioma cells were obtained by CD133 immunomagnetic kit.Transwell invasion migration assay and fluorescent matrigel degradation assay confirmed that Notch1 could promote the invasion and migration ability of GICs.It was also evaluated that Notch1 could maintains the self-renewal capacity of glioma initiation cells through sphere formation assay and in vitro limiting dilution assay.3.Notch1 regulates the invasion,migration and self-renewal abilities of glioma initiating cells through AKT/m TOR pathway mediated by CXCR4/CXCL12 system.Western blot assay found that knocking down expression of Notch1 could down regulate the protein level of Hes1,CXCR4,p AKT and pm TOR,proving CXCR4 and AKT are the downstream of Notch1 pathway.After adding CXCL12 to the control group,there was no significant change in Notch1 and Hes1 protein,but the expression of p-AKT and p-m TOR protein increased significantly,suggesting that the CXCL12/CXCR4 chemotactic factor system was downstream of the Notch1 signaling pathway in the GICs,and its activation could improve the activity of the downstream AKT/m TOR signaling pathway.4.In vivo experiments have shown that Notch1 regulates the invasion and migration of glioma initiating cells through CXCR4.The glioma initiating cells from different treatment groups were Intracranial xenografting into the nude mice.The weight and survival time of mice were observed and recorded.The size of the tumor growth was observed by the bioluminescence animal in vivo imaging system.Immunohistochemical staining was used to observe the changes in the expression of Notch1,CXCR4,p AKT and other proteins in the tissue specimens of different treatment groups.HE(hematoxylin and eosin)staining was performed to distinguish the tumor tissue form normal brain tissue.And whether the weight and survival time of the mice in different treatment groups were different.The results showed that knockdown of Notch1 can significantly slow tumor growth and improve the prognosis of mice.In the in vivo microenvironment,the Notch1-CXCR4-AKT axis pathway remains existConclusion: 1.Notch1 signaling pathway is strongly activated in glioblastoma and Notch1 is highly expressed in glioma initiating cells.2.Notch1 could promote the invasion,migration and self-renewal ability of GICs.3.Notch1 promoted the invasion,migration and self-renewal ability of GICs through AKT/m TOR pathway mediated by CXCR4/CXCL12 system.4.In vivo study demonstrated similar results consistent with the in vitro study that Notch1 regulate the biological behavior of GICs mediated by CXCR4. |
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