Function Investigation Of NT5DC2 In Glioma Stem Cell

Gliomas are the most common type of tumors involving in the central nervous system.Classified by the World Health Organization grading system,gliomas are divided into four histopathologic grades named I-IV.WHO Grade IV gliomas,also called glioblastoma(GBM),are the most aggressive brain tumor.Despite aggressive therapies,the prognosis of patients with GBM is dismal.Glioma stem cells(GSCs)are a small population of glioma cells with the characters of stem cells,such as self-renewal,differentiation potential and tumorigenesis.Recent studies suggest that GSC is a critical factor in glioma recurrence and resistance to radiotherapy and chemotherapy.Accordingly,it is of great significance for the treatment of glioma to explore the mechanism through which GSC performs its function.Our lab is devoted to studying the functions and molecular mechanism of biomacromolecules in GSCs.Based on our previous screening results on the Gene Expression Omnibus(GEO)database,we identified a serious of genes that highly expressed in GSCs relative to the bulk tumor cells(BTCs).Among these candidates,we found that NT5DC2,a functional unknown protein,was more abundant in GSCs than in non-stem tumor cells(NSTCs).Furthermore,through the analysis of TCGA database,we found that the expression of NT5DC2 in GBM was significantly higher than that in normal brain tissue,and strongly increased with glioma grades.Importantly,we found that high level of NT5DC2 significantly correlated with worse prognosis of all glioma patients and GBM patients.Therefore,NT5DC2 may play crucial roles in glioma stem cells and glioma tumorigenesis.Based on these results,this work aims to reveal the role of NT5DC2,a function unknown protein,in GSCs and glioma.Using real-time quantitative PCR and Western blot,we found that the m RNA and protein level of NT5DC2 in GSCs were much higher than that in NSTCs,and the expression of NT5DC2 gradually decreased during the differentiation of GSCs.With the immunohistochemical staining of human glioma tissue microarray,we found that NT5DC2 expression in high-grade glioma(grade III and IV)was significantly higher than that in lowgrade glioma(grade I and II).Using lentiviral infection system and sh RNA knockdown technique,we knocked down NT5DC2 in GSCs and found that deletion of NT5DC2 resulted in a strong suppression of cell viability and sphere formation of GSCs.To assess the functional consequences of NT5DC2 knockdown on GSC tumorigenic potential in vivo,we examined the tumor initiation capacity of GSCs in the orthotopic transplantation in nude mice.we found that deletion of NT5DC2 compromises the GSC tumor initiation in vivo and extends animal survival.Further,using Annexin V Apoptosis Detection Kit and flow cytometry analysis,we found that knockdown of NT5DC2 increased the apoptosis of GSCs.In summary,through a series of experiments,we found that the protein and m RNA content of NT5DC2 in GSCs were in relative high level;the high level of NT5DC2 was conducive to the maintenance of the cell viability,sphere-formation,self-renewal and tumorigenesis of glioma stem cells,but the maintenance of the cell viability of NSTCs was limited;the high level of NT5DC2 helps to inhibit the apoptosis of glioma stem cells.In conclusion,NT5DC2 plays an important role in the life activities of GSCs.Therefore,NT5DC2 has the potential to be a novel therapeutic target for GSCs.In addition,we also constructed glioma stem cells with overexpression of NT5DC2 for further mechanism investigation.In our published research,we further explored the mechanism of NT5DC2 to exert these functions through transcriptome sequencing technology,in order to provide support for the development of drugs and therapeutic strategies for NT5DC2.