The Effect Of WNK3 On Autophagy Of Glioma Cells Induced By Temozolomide

Objective: Human glioma is derived from glial cells and is a common primary tumor in the central nervous system.It has a high degree of malignancy and can quickly spread to brain tissue far from the primary tumor,and is deeply infiltrated with surrounding brain tissue.Therefore,it is difficult to completely remove the glioma tissue.At present,the standard treatment methods of glia all over the world are surgical resection,postoperative concurrent radiotherapy and chemotherapy and other comprehensive treatment methods.In the latest World Health Organization(WHO)classification of central nervous system tumors(2016),molecular biomarkers are combined with typical histological features for the first time to define different gliomas.The new diagnostic method of glioma indirectly indicates a deeper understanding of the biological behavior of brain tumors.At the same time,more and more attention has been paid to individualized treatment of glioma patients,which may become a breakthrough in glioma treatment in the future.The subfamily of WNK protein kinase consists of four genes,WNK(1-4),which is characterized by a typical sequence variation in the conserved catalytic domain,named for the lack of lysine in its functional domain.Although most studies have focused on the role of WNK1,WNK3 and WNK4 in regulating different ion transporters in the kidney and extrarenal tissues,there is growing evidence that WNK kinases play other roles in various signal cascades associated with cancer.They play an important role in the progression of G1/S cell cycle,the adaptation of metabolic tumor cells,and the escape of apoptosis and metastasis.However,the study of WNK in autophagy phenotype of glioma cells is still blank.Methods:In the experiment,we first detected the expression of WNK3 in glioma tissues by immunohistochemistry,and also detected the expression of WNK3 in three commonly used glioma cell lines U87,U251 and U373 by Western blot method.Then,we constructed the related plasmids to silence the expression of WNK3 in U87 cells,and used Western blot to detect whether the WNK3 gene was silenced.Next,we performed cytological experiments using WNK3-silent U87 cells.CCK-8 method was used to detect the effect of the same concentration of TMZ on different groups,and to observe whether there was significant difference in cell survival rate.At the same time,autophagy associated protein LC3 B,P62,Beclin1 was detected by Western blot.Then,the ultrastructural changes of glioma cells in different groups treated with TMZ were observed by transmission electron microscope(TEM).Results:The results showed that the expression of WNK3 was expressed in U87,U251 and U373 cell lines.The expression level of WNK3 in advanced gliomas(Grade III and IV)was significantly higher than that in low-grade gliomas(Grade I and II).After silencing WNK3,the expression of autophagy associated protein in U87 treated with TMZ was lower than that in negative control by WB.We also found that the bilayer membrane structure and autophagy lysosomes containing residual organelles in shRNA-WNK3 U87 cells treated with TMZ were less than those in the negative control group.Conclusion: through the above experimental results,it can be concluded that silencing WNK3 can reduce the autophagy of glioma cells induced by TMZ,and may change the drug resistance of glioma to chemotherapeutic drug TMZ through autophagy.It may be a target in the future direction of glioma treatment.