NKCC1 Promotes Epithelial-mesenchymal Transition-like In Glioblastoma

Glioblastoma is the most common and lethal primary intracranial tumor.As the key regulator of tumor cell volume,NKCC1 expression increases along with the malignancy of the glioma,and NKCC1 has been implicated with glioblastoma invasion.However,little is known regarding the role of NKCC1 in the epithelial-mesenchymal transition-like process in gliomas.We noticed that aberrantly elevated expression of NKCC1 leads to changes in the shape,polarity and adhesion of cells in a glioma.Here,we investigated whether NKCC1 promotes an EMT-like process in gliomas.Pharmacological inhibition and knockdown of NKCC1 both decreased the expressions of mesenchymal markers,such as N-cadherin,vimentin and snail,whereas these treatments increased the expression of the epithelial marker E-cadherin.These findings indicated that NKCC1 promoteed an EMT-like process in gliomas.The underlying mechanism is the facilitation of the binding of Rac1 and RhoA to GTP by NKCC1,which results in a significant enhancement of the EMT‐like process.Specific inhibition or knockdown of NKCC1 both attenuate activated Rac1 and RhoA,and the pharmacological inhibitions of Rac1 and RhoA both impair the invasion and migration abilities of gliomas.These findings suggested that elevated NKCC1 activity acts in the regulation of an EMT-like process in gliomas and thus provide a novel therapeutic strategy for targeting the invasiveness of gliomas.This study including four parts: 1.To detect the expression of NKCC1 in gliomas.Fifty-two glioma samples with different malignancy were obtained from surgey for tissue microarray immunohistochemical staining.Western blot was used to detect NKCC1 expression in seven human malignant glioma cell lines,and the cell lines with higher expression of NKCC1 were selected for later experiments in vitro experiments.The results showed that,NKCC1 expression in GBMs patients who has formed multiple regional small satellite lesions,was significantly higher than other GBM patients had relatively circumscribed margins.We demonstrated that the NKCC1 expression was elevated in GBM by immunohistochemical staining.The results of western blot were also consistent with immunohistochemical results.2.To investigate the role of NKCC1 in migration and invasion of glioma cells.In U87 and SNB19 glioma cell lines,the expression of NKCC1 protein was increased significantly,which was selected for experiments in vitro.In normal serum culture conditions,shRNA aimed at silencing NKCC1 was transfected into glioma cell line to knock down the NKCC1 expression.Those cells were assigned into three group:shNKCC1-1 group,shNKCC1-2 group and scrambled group.And bumetanide was treated to glioma cell lines,made it into three groups: control group、BMT(10μΜ)group and BMT(100μΜ)group.The expression of NKCC1 was detected at protein level by western blotting,transwell invasion assay and wound healing assay were performed to determine the migration and invasion of glioma cells.The results showed that the ability of migration and invasion of glioma cells was markedly impeded b y inhibiting NKCC1.3.To investigate the role of NKCC1 in EMT in glioma cells.In normal serum culture conditions,shRNA aimed at silencing NKCC1 was transfected into glioma cells to knock down the NKCC1 expression,and bumetanide was used to inhibit NKCC1.The expression of EMT related protein,including E-cadherin,N-cadherin,vimentin and snail was detected at protein level by western blotting and immunofluorescence staining.The results showed that the expression of the epithelial marker E-cadherin increased with the drug concentration,and the expressions of mesenchymal markers,such N-cadherin,vimentin and snail,gradually decreased.We also demonstrated that the expression of the epithelial marker E-cadherin increased and the expressions of mesenchymal markers,such N-cadherin,vimentin and snail decreased in NKCC1 konwed-down group.4.Rac1 and RhoA signaling mediated NKCC1-induced EMT.The TCGA data set was searched to demonstrate that the expression of the SLC12A2 gene was positively correlated with the expressions of the RAC1 and RHOA genes.We performed GST-TRBD and GST-PBD pull-down assay,and RhoA and Rac1 activities were decreased basally following BMT treatment in both the U87-MG and SNB19 cells.EGF stimulation increased the activations of RhoA and Rac1,while treatment with BMT attenuated this activation.Next,we evaluated the activations of RhoA and Rac1 in NKCC1 knockdown U87-MG and SNB19 cells,and an effect similar to that of BMT was observed.Finally,to implicate these downstream pathways in the regulation of the migration of glioma cells,we treated U87-MG and SNB19 cells with small molecule inhibitors of Rho-associated kinase(Y27632)and Rac1(NSC23766)and assessed the effects on migration using a Transwell migration assay.We found that both compounds significantly reduced the migration of U87-MG and SNB19 cells.Furthermore,we simultaneously treated U87-MG and SNB19 cells with BMT(100 μM)and small molecule inhibitors of Rho-associated kinase(Y27632)or inhibitors of Rac1(NSC23766).In a similar manner,we applied Y27632 or NSC23766 to NKCC1 knockdown cells or scrambled cells to evaluate the migration ability via the Transwell assay,and the results revealed that there was no significant difference between the combined inhibition and the single inhibition of NKCC1.Taken together,these findings suggest that NKCC1 is required for the activation of RhoA and Rac1.Conclusion: 1.Overexpressed NKCC1 drove multifocal tumor infiltration and spread,and was correlated with shorter survival in GBM;NKCC1 is expressed in glioma tissues at a higher level in GBM compared with lower grade gliomas.2.Inhibition of NKCC1 decreased invasion and migration of glioma.3.NKCC1 promoted EMT in U87-MG and SNB19 cells.4.Rac1 and RhoA signaling mediated NKCC1-induced EMT.