The Role Of PIK3CD In Human Gliomas Induced Angiogenesis And Its Mechanism

Malignant glioma is the most common and lethal primary solid tumor in adult central nervous system.Gliomas are classified into four grades according to their pathological characteristics(The World Health Organization standards).Low grade gliomas(Grade II)could progress to high grade ones(Grade III and IV).Malignant gliomas are difficult to be completely resected with high relapse rate.Glioblastoma(GBM)is the most malignant glioma and the 5-year-survival rate is very low.The mean survival of GBM patients is 12 to 15 months.The existence of abundant and heterogeneous blood vessels is a typical pathological feature of GBM,which promote tumor cell proliferation,invasion and recurrence.Therefore,investigation of the mechanisms underlying glioma angiogenesis is important to develop targeting therapeutic strategies and improve treatment efficiency.Recent studies have shown that multiple gene mutation,deletion and abnormal expression,including P53,PTEN,IDH1 and PI3 K,are crucial for glioma initiation and progression.Previous studies have demonstrated that the aberrant activation of PI3K/Akt signaling pathway in glioma is closely associated with tumorigenesis,progression,invasion and recurrence.We previously found that PI3K/Akt signaling pathway play an important role in glioma stem cell-mediated tumor angiogenesis.The class IA phosphatidylinositol 3-kinases(IA PI3Ks)are composed of the regulatory subunit p85 and the catalytic subunit p110.The catalytic subunit p110 of classic IA PI3 K has three isoforms,i.e.,p110?,p110?,and p110?.The expression and distribution of p110 isoforms are different in tissues and organs.While p110? subunit and p110? subunit are widely expressed in tissues,p110? subunit only expresses in the immune system and a few types of tumors.P110? is encoded by PIK3 CD and can constitute with p85 regulatory into PI3 K to exert biological effects.In this study,we investigated the role of PIK3 CD in glioma angiogenesis and the underlying mechanisms.In the first part,we examined the expression and distribution of PIK3 CD in glioma specimens using immunohistochemical staining,and analyzed the correlation between PIK3 CD expression and clinical pathological parameters in patients of glioma.In the second part,we disrupted PIK3 CD expression in human glioma cell lines using lentiviral shRNA vectors.We detected the effects of silencing PIK3 CD expression in gliomas on the proliferation,migration,and tube formation of the endothelial cells using in vitro co-culture system.In the third part,we established glioma xenograft model to investigate the role of PIK3 CD in angiogenesis and the potential mechanism in vivo.The effect of PIK3 CD disruption on the survival of tumor-bearing mice and microvessel density of the transplanted tumor were determined.Collectively,this study includes the following three parts:1.The relationship between PIK3 CD expression and clinical pathological parameters in glioma patients.We discussed the correlation between the expression of PIK3 CD and clinic pathological features in 166 human gliomas.P110?,which is encoded by PIK3 CD,is mainly expressed in the cytoplasm of glioma cells.We only found a little expression of PIK3 CD in the tumor cell nucleus and almost no expression in the tumor blood vessels.The expression of PIK3 CD is correlated with glioma grades.In high grade gliomas(WHO grade III and IV),PIK3 CD high-expressed patients proportion is large(34% and 46%),and in low grade glioma patients(WHO grade II),the proportion accounted for PIK3 CD high-expressed patients is small(5%).In the grade I glioma,PIK3 CD expression is almost undetectable.We also found that PIK3 CD level predicted poor prognosis of patients with gliomas using Kaplan-Meier analyses.Patients in PIK3CD-high group were significantly shorter in overall survival time.In addition,we found that there was a positive correlation between the expression of PIK3 CD and MVD in glioma specimens.2.The promoting effect of PIK3 CD on angiogenesis in vitro.Using lentiviral interference method,we knocked down PIK3 CD expression in human glioma cell lines.By real-time quantitative PCR and Western Blot,we verified the reduced expression of PIK3 CD in glioma cells transfected with PIK3CD-shRNA.We then used co-culture system to determine the role of the supernatant secreting by PIK3CD-silencing glioma cells on endothelial cells proliferation,migration,and tube formation.The promoting effect was attenuated compared with the control group.We also overexpressed PIK3 CD in glioma cells.Through co-culture technology,we found over-expressed PIK3 CD in glioma cells can promote endothelial cell proliferation,migration,and tube formation.Mechanically,disrupting PIK3 CD expression in glioma cells impaired the secretion of VEGF and vice versa.These results were confirmed by ELISA,showing that PIK3 CD controlled VEGF production from glioma cells.3.Interfering PIK3 CD in glioma orthotropic transplantation model.Using lentiviral interference technique,we disrupted PIK3 CD expression in a human glioma cell line(U87)and constructed xenografts using PIK3CD-silencing tumor cells.Compared with the control group,the survivals of the mice bearing PIK3CD-silencing xenografts were prolonged.Moreover,the tumor vessels in PIK3CD-silencing xenografts were reduced relative to the control ones.Taken together,our study demonstates that overexpressed PIK3 CD in human gliomas promotes tumor angiogenesis through inducing VEGF expression.