PDCL3 Regulates VEGF/VEGFR-2 Signal Pathway Mediates Glioma-associated Mesenchymal Stem Cells Promoting Angiogenesis In Glioma

Objective: As an important part of glioma microenvironment,mesenchymal stem cells play an important role in the development and invasion of glioma.Recent studies have shown that glioma-associated mesenchymal stem cells can transform into vascular pericytes under specific conditions,thus promoting angiogenesis.However,its biological characteristics and related molecular mechanisms and signal pathways have not been fully understood.Therefore,we use specific conditions to simulate the glioma microenvironment in order to study the mechanism of mesenchymal stem cells in tumor development.Methods: Human glioma-associated mesenchymal stem cells were isolated and extracted from human glioma specimens.In vitro experiment: mesenchymal stem cells were treated with different conditioned media and transwell co-culture respectively;the expressions of m RNA and lnc RNA in different treatment groups were detected by gene chip technology,and the target gene prediction and pathway enrichment analysis were performed;the corresponding gene expression in human glioma samples was detected by immunohistochemistry and immunofluorescence. Mesenchymal stem cells were transfected with viral vector to knock down the target gene,and the cell viability was detected by CCK-8;the target gene expression of mesenchymal stem cells after different treatments was detected by western-blot technique;and the angiogenic ability of mesenchymal stem cells with different treatments was detected by angiogenesis experiment.In vitro experiment: glioma cell line U87 and human glioma-associated mesenchymal stem cells were mixed and inoculated into the brain of 4-6-week-old nude mice,and the survival of nude mice was recorded.The tumor tissue was removed 28 days later,and the expression level of target gene in tumor tissue was detected by immunohistochemistry.Results: In vitro experiment,human glioma-associated mesenchymal stem cells underwent morphological changes after co-culture with transwell and conditioned medium.Gene chip detection of mesenchymal stem cells showed that the expression of angiogenesis-related target gene PDCL3 was significantly increased,western-blot detection of PDCL3 expression was significantly increased,and pathway enrichment analysis found that it was involved in the expression of VEGF/VEGFR-2 signal pathway.Immunohistochemistry and immunofluorescence detection showed that the expression of PDCL3 and VEGFR-2 was increased near the blood vessels,and the expression of α-SMA and CD31,pericyte markers related to the transformation of human glioma-related mesenchymal stem cells and vascular endothelial cell,were also increased.After virus transfection,the high expression of GFP tag was detected by fluorescence microscope,and the expression of PDCL3 was observed decreased by western-blot.Angiogenesis experiment showed that after knocking down PDCL3,the tube-forming ability of mesenchymal stem cells was significantly weaker than that of the control group.In vitro experiment,immunohistochemical staining of mouse brain tissue showed that PDCL3 and VEGFR-2 were highly expressed around the lumen.Conclusion: In glioma microenvironment,the increased expression of PDCL3 gene in human glioma-associated mesenchymal stem cells affects the expression of downstream VEGF/VEGFR-2 pathway and promotes angiogenesis.After knocking down the expression of PDCL3,the ability of mesenchymal stem cells to form tubes was significantly weakened and the ability of angiogenesis was inhibited.