Molecular Mechanism Of GBP2 Promoting Glioma Proliferation

Objective:Glioma is the most common primary malignant tumor of the central nervous system,with poor treatment effect and poor prognosis.To clarify the pathogenesis of glioma is the key to its early diagnosis and treatment.Guanylate binding protein 2(GBP2)plays an important role in the development of several cancers.Our previous studies showed that GBP2 was significantly overexpressed in glioma samples.GBP2 knockdown can inhibit the proliferation of glioma cells,while GBP2 overexpression can promote the proliferation of glioma cells.This study was aimed to explore the molecular mechanism of GBP2 promoting glioma proliferation.Methods:Gene microarray was performed to search for differentially expressed genes after GBP2 depletion.IPA analysis was performed base on the microarray data.The network analysis indicated that EGFR as the target of GBP2.Western blotting was used to detect the levels of key proteins in the EGFR pathway in U251 and U87 glioma cell lines.Mass spectrometry and co-IP were used to search for proteins directly interacting with GBP2.The results indicated that GBP2 could interact with KIF22 in glioma cells.CCK8 assay,clone formation assay and Flow cytometry were performed to detect the effect of KIF22 knockdown on the cell viability,clone formation ability and the cell cycle of glioma cells,respectively.Plasmids were constructed for knockdown of GBP2 and overexpression of KIF22 at the same time and were transfected into U87 and U251 cells.Western blotting,CCK8 assay and clone formation assay were performed to determine whether GBP2 regulated EGFR expression level and proliferation ability of glioma cells through KIF22.Results:1.The results of Gene microarray,bioinformatics analysis and Western blotting showed that EGFR was the target of GBP2.After GBP2 knockdown,the expression levels of EGFR and phosphorylated EGFR in glioma cells decreased significantly while GBP2 overexpression effectively increased the expression of EGFR and P-EGFR,suggesting that GBP2 regulated the activity of EGFR pathway.2.The results of mass spectrometry combined with co-IP showed that GBP2 could interact directly with KIF22.The results of Western blotting showed that the expression of KIF22 decreased after GBP2 knockdown in U87 and U251 cell lines.In U87 cell line,GBP2 overexpression increased the expression level of KIF22.These results indicate that GBP2 interacts with KIF22 and regulates the expression of KIF22.3.The results of CCK8 assay,clone formation assay and Flow cytometry showed that the cell viability and clone formation ability of U251 and U87 cells were significantly decreased,while the cell cycle was arrested in G0/G1 phase after KIF22 knockdown.These results indicate that knockdown of KIF22 inhibited the proliferation of glioma cells in vitro.4.Down-regulation of EGFR and P-EGFR expression induced by GBP2 knockdown was reversed by overexpression of KIF22.The decrease in the cell viability and clone formation ability after GBP2 knockdown was reversed by KIF22 overexpression.These results indicate that GBP2 regulates EGFR signaling pathway through KIF22 and promotes glioma proliferation.Conclusion:GBP2 was highly expressed at the m RNA level in glioma cells by RT-q PCR.After knocking down the expression of GBP2 in glioma cell lines,the proliferation of glioma cells was inhibited,and GBP2 was overexpressed on the contrary.Then,it will promote the proliferation of glioma cells.It is suggested that the overexpression of GBP2 is positively correlated with the occurrence and development of glioma,and reducing the expression of GBP2 can inhibit the proliferation of glioma cells.This result can provide new valuable therapeutic directions and ideas for the treatment of glioma.