Study On The Role Of NHE1 In Invasion And Migration Of Glioma Cells

Glioblastoma is the most common primary tumor in central nervous system,with the characteristics of highly proliferation,abnormal angiogenesis and invasion and migration to the surrounding areas.Despite the continuous improvement of new imaging technologies and development of surgery,the extent of resection for gliomas has been greatly increased and the patients' median survival was prolonged.However,due to the characteristics of invasion and migration,the patients might face inevitable relapse and poor prognosis.Tumor microenvironment is a complex dynamic local environment composing of tumor cells,stromal cells(immune cells,fibroblasts,etc.),extracellular matrix and a variety of extracellular factors which plays an important role in tumor cell development,proliferation,and invasion and migration.Rapid proliferation of tumor cells,abnormal blood vessels and abnormal energy metabolism would result in tumor cells nutrition deficiency and acid extracellular environment as well.In order to evade this environmental stress,migration to relatively nutrient rich region might be an optimal choice to survive for tumor cells.Sodium hydrogen exchanger 1(NHE1)is a member of sodium hydrogen exchangers family,which is widely expressed in cell membrane of various type of cells.In physiological condition,NHE1 plays a critical role in intracellular pH(pHi)and cell volume homeostasis.NHE1 is also an important regulator in extracellular acidification and upregulated or activated in a variety of tumors and leads to invasion and migration,such as hepatocellular carcinoma,cervical carcinoma,breast cancer and so on.However,the role of NHE1 remains unclear in glioma.This study aims to investigate the expression of NHE1 in gliomas and the underlying mechanisms of NHE1 in invasion and migration of glioma cells,including two parts:1.To detect the expression of NHE1 in gliomas and analyze the relationship with clinical prognosis.First of all,we downloaded the gene expression data and clinical data of patients with GBM and lower grade glioma patients(LGG),including grade WHO?-? in TCGA database and analyzed the expression of NHE1,statistical assessment was performed for the relationship between NHE1 expression and prognosis in GBM patients.In addition,40 glioma samples with different pathological grades and 10 control brain tissue samples were obtained from surgey for immunohistochemical staining;At the same time,Real-time PCR and western blot were employed to detect the expression of NHE1 at mRNA level and protein level.Then,western blot was used to detect the expression of NHE1 in four human malignant glioma cell lines,and the cell lines with higher expression of NHE1 were selected for the later in vitro experiments.The results showed that NHE1 expression in 172 GBMs was significantly higher than that in 350 LGGs,prognosis of patients with GBM was negatively correlated with the expression of NHE1.The immunohistochemical staining demonstrated that the NHE1 expression was elevated in GBM,and the expression level increased positively with tumor pathological grade.The results of Real-time PCR and western blot were also consistent with immunohistochemical results.In U87 and SNB19 glioma cell lines,the expression of NHE1 protein was increased significantly,which was selected for in vitro experiments.2.To investigate the role of NHE1 in glioma cell migration and invasion.In low serum culture conditions,siRNA aimed at silencing NHE1 was transfected into U87 and SNB19 cell lines to knock down the NHE1 expression.Those cells were assigned into three group:siRNA-NHE1 group,siRNA-NC group and control group.The expression of NHE1 was detected at mRNA and protein level respectively using Real-time PCR and western blot,transwell invasion assay and wound healing assay were performed to determine the migration and invasion of glioma cells,the expression of MMP-2,MMP-9,cortactin and p-cortactin were detected by western blot,the invadopodia and NHE1 location of glioma cells were observed by immunofluorescence,the formation of invadopodia and the invasion ability were detected by fluorescent Matrigel degradation experiments.The results showed that NHE1 was enriched in cell barbed end of invadopodia,compared with control group and siRNA-NC group.The expression of NHE1 was inhibited at both mRNA and protein level after transfected,the ability of migration and invasion of glioma cells was markedly impeded.In the siRNA-NHE1 group the expression of MMP-2,MMP-9 and p-cortactin were decreased significantly,while cortactin did not change significantly,the invadopodia formation and the degraded area of matrix for each of the tumor cells also decreased significantly.Conclusion:1.Expression of NHE1 in glioma tissues compared with control brain tissue,and the expression is significantly increased in GBM tissue than that in lower grade glioma tissue,the expression is negatively correlated with the prognosis of patients with GBM.2.Knocking down NHE1 expression can inhibit the expression of MMP-2 and MMP-9 in glioma cells and decrease formation of invadopodia and ability of invasion.3.NHE1 is enriched in cell barbed end of invadopodia,which might affect the formation of invadopodia by altering the local pH of invadopodia.4.Targeting at NHE1 is expected to inhibit the invasion and migration of glioma cells.