Mechanism And Effect Of OLFML2A On Glioblastoma Cells Senescence Through Notch Signaling Pathway

Objective: By exploring the effect of OLFML2A on glioblastoma cell senescence and its mechanism,this study could provide potential therapeutic targets for the diagnosis,pathological grade and precision treatment of glioblastoma.Methods: 1.The expression and clinical significance of OLFML2A and the cell senescence markers p53,p21 and p16 in glioblastoma were investigated by analysing relevant data from TCGA and CGGA databases and using Western Blot and immunohistochemical staining experiments in glioma tissues.2.The effect of OLFML2A on cell senescence and senescence-associated secretory phenotype of glioblastoma cells was investigated by analyzing relevant data from TCGA database and using qRT-PCR,Western Blot,senescence-associated β-galactosidase staining and ELISA in glioblastoma cell lines U87-MG and U251 with OLFML2A gene knockdown.3.The effect of OLFML2A on the Notch signaling pathway in glioblastoma cells was investigated by analyzing the results of gene sequencing in glioma cells with OLFML2A gene knockdown and using qRT-PCR and Western Blot in glioblastoma cell lines U87-MG and U251 with OLFML2A gene knockdown.4.The mechanism of OLMFL2A on cell senescence in glioblastoma cells was investigated by using DAPT,a Notch signaling pathway inhibitor,and qRT-PCR and Western Blot in the glioblastoma cell line U87-MG with OLMFL2A gene knockdown.5.The mechanism of OLMFL2A on cell senescence in glioblastoma cells was demonstrated by constructing the nude mouse intracranial orthotopic tumor model and using Western Blot and immunohistochemical staining.Results : The result of bioinformatics analysis,Western Blot and immunohistochemical staining showed that compared with normal tissuess,OLFML2A and cell senescence markers p53,p21,p16 was highly expressed in glioblastoma,and were positively correlated with the pathological grade of glioma.2.The result of bioinformatics analysis showed that the expression of OLFML2A in glioma cells was positively correlated with the expression of cell senescence markers p53,p21,p16 and senescence-associated secretory phenotypically molecules IL-6,IL-1β.The result of qRT-PCR,Western Blot,senescence-associatedβ-galactosidase staining and ELISA showed that the expression of cell senescence markers p53,p21,p16 and senescence-associated secretory phenotypically molecules IL-6 and IL-1βwas significantly decreased in glioblastoma cell lines U87-MG and U251 with OLFML2A gene knockdown.3.The result of qRT-PCR and Western Blot experiments showed that the expression of Notch1,Notch2 and Hes1,key proteins of Notch signaling pathway,were significantly decreased in glioblastoma cell lines U87-MG and U251 with OLFML2A gene knockdown.4.The result of qRT-PCR and Western Blot showed that using Notch signaling pathway inhibitor DAPT in glioblastoma cell lines with OLFML2A gene knockdown could reverse the decrease expression level of the cell senescence markers p53,p21 and p16 caused by OLFML2A gene knockdown.5.The result of Western Blot and immunohistochemical staining showed using Notch signaling pathway inhibitor DAPT in glioblastoma cell lines with OLFML2A gene knockdown could reverse the decrease expression level of the cell senescence markers p53,p21 and p16 caused by OLFML2A gene knockdown in the nude mouse intracranial orthotopic tumor model.Conclusion : Downregulation of OLFML2A gene expression inhibits glioblastoma cell senescence by activating the Notch signaling pathway in animal models and cells.