The Roles Of Pirin On The Proliferation And Self-renewal Of Glioma Stem Cell

Glioma is a type of prevalent and malignant brain tumor with gial cell characteristics.Glioblastoma multiforme(GBM)is the most malignant primary brain tumor in central nervous system.The characteristics of infiltrative nature make it impossible to remove the tumor completely by surgery.Clinically,surgery,chemotherapy and radiation therapy usually used for combination treatment,but the cancer usually recurs.The most common median survival following diagnosis is 12 to 15 months despite maximum treatment.Glioma stem cell(GSC)has the characteristics of stem cells such as self-renewal and differentiation potential.GSC is resistant to radiotherapy and chemotherapy,GSC contributes to tumor initiation and therapeutic resistance.GSC not only actively remodel the tumor microenvironment,but also receive critical factors to maintain its survival.Most conventional anti-tumor treatments target proliferating cells.In order to eliminate GSC effectively,it is particularly important to target its interaction with the tumor microenvironment.Drugs target secreted proteins directly in the tumor microenvironment,which can obviate the need for drug to move intracellularly.It is of great significance to find secreted proteins that can serve as targets for the treatment of gliomas.SWATH-MS is an advanced label-free quantitative method,using data independent acquisition mode and targeted data extraction strategy,which can used for low abundance protein,with high quantitative coverage,high quantitative accuracy,and simple sample preparation.A quantitative proteomic study of the culture supernatants of GSC and its corresponding non-stem tumor cells(NSTC)was performed using SWATH-MS.A total of 647 proteins were reliably quantified and 58 were significantly changed.39 proteins were high in the GSC culture supernatant and 46 proteins were low.The clinical correlation analysis of a series of differential proteins was performed using a database of brain tumors.We found that the expression of Pirin in GBM is higher than normal brain tissue.The prognosis of glioma with high Pirin expression is significantly poorer.We identified Pirin as a further study object to study the function and regulatory mechanism of Pirin in GSC.First,Western Blot and Real-time PCR were used for detect the change of Pirin expression after GSC differentiation.The results showed that Pirin was highly expressed in GSC specifically at the protein level and mRNA level.During GSC differentiation,Pirin protein expression levels gradually decreased.To investigate whether Pirin plays a specific role in GSC,we constructed three Pirin shRNA plasmid to stably knock down the expression of Pirin in GSC by using lentivirus infection system.The results showed that knocking down the expression of Pirin could significantly inhibit the ability of GSC to form tumor sphere and the viability of GSC with no significant effect on the viability of NSTC.With the chemotherapy drug temozolomide(TMZ)as a positive control,Pirin specific inhibitor Triphenyl compound A(TPh A)could also significantly inhibit the viability of GSC.In summary,we have established a quantitative proteome analysis method for glioma and found that Pirin is highly expressed in GSC,the prognosis of glioma patients with high Pirin expression is significantly poorer.Knockdown Pirin significantly inhibited the ability of self-renewal and proliferation in GSC,but not NSTC,suggesting that Pirin may specifically function in GSC.Next,we will establish orthotopic xenografts in immunocompromised nude mice to determine the functional consequences of Pirin knockdown in vivo.To explore the molecular mechanism of Pirin in GSC,Pirin-Flag plasmids constructed were used for stably overexpress Pirin in GSC.Western blot results show that the Pirin-Flag plasmid can be correctly expressed in GSC,we have established the stable GSC cell line with Pirin-Flag overexpressed.In the following study,in order to reveal the specific regulatory mechanism of Pirin on GSC,we will search for proteins that interact with Pirin in GSC through immunoprecipitation and mass spectrometry analysis.We hope to provide new ideas and strategies for the treatment of clinical glioma patients.