WNK3 Promotes The Invasiveness Of U87 Cells Under Hypoxia By Inducing The Epithelial To Mesenchymal Transition

Objective: Glioma,which originated from glial cells,is the most common intracranial malignant tumor in the adult brain.The most malignant tumor type is glioblastoma multiforme(GBM).Due to the high degree of malignancy,glioma often infiltrates the surrounding brain and the demarcation among normal tissue is unclear,so that is difficult to be completely removed by surgery.At present,the average survival time is only 14 months with the combined treatment of surgery,standard radiotherapy and chemotherapy.Changes of individual gene phenotype in glioma patients may be a breakthrough for future treatment.With-No-Lysine Kinase(WNK)is a member of the serine / threonine protein kinase family;it is named for the absence of a conserved lysine within the kinase domain.The WNK family comprises four human genes,WNK1-4.WNK1,WNK2 and WNK3 have been widely involved in the process of tumor proliferation,apoptosis,invasion and tumor microenvironment.It was considered that WNK2 may be a tumor suppressor gene that is specific to the tumor of the nervous system,and WNK1 and WNK3 was involved in the invasion and apoptosis of the tumor.It is well known that extensive hypoxic necrosis and angiogenesis within GBM tumors are the most prominent histological features.Studies have shown that hypoxia may changes in tumor phenotypes,including increased metastases,tumor recurrence,resistance to chemotherapy,resistance to radiation,increased invasion,decreased patient survival,genomic instability,decreased apoptotic potential,alterations of gene expression,oncogene activation,induction of angiogenesis,gene amplification,increased brain tumor stem-cells population,and stem-cell genetic instability.However,the molecular mechanisms underlying glioma adaption in hypoxic microenvironments are poorly understood,and the mechanism of WNK kinase in the regulation of glioma under hypoxia is not clear.Methods: In the experiment,we used Western blot to detect the expression of WNK3 in U87 cells in the hypoxic environment.We used immunohistochemical analysis to evaluate the expression of WNK3 in human glioma specimens.We also used shRNA to knockout the expression of WNK3 in U87 cells and used Western blot to verify the expression.Subsequently,a series of cell biological function tests were performed using the knockout WNK3 U87 cell line.Proliferation and cell cycle assays were performed using CCK-8 assay and flow cytometry,followed by Western blot to detect the cell cycle associated protein CyclinD1.Trans-well assay was used to detect cell invasion,Western blot was used to detect MMP-2 and MMP-9,which are involved in glioma invasion.We also examined the EMT-associated proteins Snail1,Vimentin and E-cadherin using Western blot.Results: Our results showed that WNK3 expression was significantly higher in U87 cells under hypoxic environment and remarkably higher in high-grade(III and IV)gliomas than in low-grade(I and II)gliomas.In addition,WNK3 knockdown was shown to inhibit the invasion of U87 glioma cells through the regulation of the epithelial-to-mesenchymal transition,especially under hypoxic conditions.The results of detected invasion-related proteins and EMT-related proteins are consistent with the experimental results.We also found that knockout of WNK3 in U87 cell lines reduced cell proliferation and induced cell cycle arrest.The results of detection of cell cycle related protein were consistent with the experimental results.Conclusion: Taken together,WNK3 plays an important role in the hypoxic microenvironment of gliomas and might be a candidate for therapeutic application in the treatment of glioma.