The Function And Molecular Mechanisms Of Prrx1 In The Stem-like Traits And Angiogenesis Of Human Glioma

BackgroundGlioma is the most common malignant tumor of the central nervous system and the current treatment is mainly surgical resection.However,it is difficult to be radical resected by surgery due to the extensive infiltrative growth characteristic of glioma.Therefore,it is necessary for postoperatives to receive further comprehensive treatment,such as radiotherapy,chemotherapy,targeted therapy,immunotherapy,etc.In spite of the aforementioned therapies,the median survival time is still only 14.6 months,with a very high recurrence rate and mortality.Accumulating evidences have indicated that Glioma Stem Cells(GSCs)and the GSCs-mediated excessive angiogenesis may be the key bottleneck restricting the curative effect and prognosis of glioma patients.In this study,the candidate gene Prrx1,which was significantly upregulated in glioma and was closely related to glioma stemness as well as angiogenesis,was screened from the expression microarray.We aimed to clarify the molecular regulatory mechanism of Prrxl in glioma,so as to provide a new perspective and strategy for improving the current treatment status of glioma patients.Objectives(1)To analyze the expression and clinicopathologic significance of Prrx1 in glioma tissues.(2)To investigate the effect of Prrx1 expression on the glioma stemness.(3)To investigate the effect of Prrx1 expression on glioma angiogenesis.(4)To elucidate the molecular mechanisms undergoing Prrx1 regulating the biological function of glioma cells.Methods(1)Prrx1,a candidate gene related to the occurrence and development of glioma,was screened out through bioinformatics analysis.RT-qPCR,IHC and GSEA were conducted to investigate the expression pattern and biological function of Prrx1 in gliomas.(2)The correlation between the Prrx1 expression level and glioma stemness was investigated by EdU,sphere formation assay,clony formation assay,western blot and RT-qPCR.The correlation between the Prrx1 expression and glioma angiogenesis was examined by tube formation assay,CAM assay,transwell,RT-qPCR and ELISA assay.(3)GSEA,western blot,dual-luciferase reporter assay,ChIP and rescue experiments were conducted to investigate the molecular mechanisms underlying Prrx1 regulating stemness and angiogenesis in gliomas.(4)Paraffin sections of tumor tissues from 74 glioma patients were collected and the orthotopic glioma transplantation model was established to verify the in vitro results above by IHC and IF assays.Result(1)The expression level of Prrx1 in glioma tissues was significantly higher than that in normal brain tissues,and its excessive expression was significantly associated with poor prognosis of glioma patients.RT-qPCR and Western blot results showed that Prrx1 positively regulate the expression levels of GSC markers(such as SOX2,etc.)and angiogenesis markers(such as VEGF-A,etc.).Phenotypic experiment showed that Prrx1 enhanced glioma stem cell-like properties and angiogenesis.(2)GSEA analysis of multiple glioma expression profiles showed that the high expression of Prrx1 was significantly correlated with the activation of TGF-?/smad signaling pathway.Western blot results showed that Prrx1 could up-regulate the phosphorylation levels of Smad2 and Smad3,key effector molecules of TGF-?/smad pathway,as well as the expression levels of TGF-?1,key upstream molecular of TGF?/smad pathway.Dual-luciferase reporter assay and ChIP assay revealed that Prrx1 could directly bind to the promoter region of TGF-?1 gene and transactivated TGF-?1 expression.The IHC staining of orthotopic xenografts and clinical glioma specimens,as well as analysis of GEO datasets further verified the aforementioned conclusions.ConclusionPrrxl is significantly upregulated in glioma tissues and associated with poor prognosis of glioma patients.As a transcription factor of TGF-?1,it directly binds to the TGF-?1 gene promoter and activates the expression and secretion of TGF-?1.The upregulated TGF-?1 acts on glioma cells in an autocrine way and activates the downstream TGF-?/smad signaling pathway,which cooperates with Prrx1 to upregulate the expression levels of GSC markers and angiogenesis markers,thus promoting the glioma stemness and angiogenesis.