The Role And Mechanism Of ZYX In Promoting Invasion And Metastasis Of Gastric Cancer And Its Role In Glioma Invasion

Gastric cancer is the leading cause of tumor-related death ranking fifth among all tumors worldwide.Despite continuous improvement of treatment strategies,the prognosis of patients with gastric cancer is still unsatisfactory.Invasion and metastasis are important biological characteristics of gastric cancer,and also the main factors resulting in poor prognosis of the patients with GC.Therefore,to explore the underlying molecular mechanisms of the invasion and metastasis is of great significance for discovering new targets thereby developing new strategies for gastric cancer treatment.Adhesion plaques play an important role in the connection between cytoskeletal proteins and the extracellular matrix and the transmission of extracellular signals into the cells.ZYX is the main component of adhesion plaques.It contains LIM domains and can act as a scaffold mediating interactions between various proteins to regulate cell adhesion,proliferation and migration.ZYX can also shuttle through the nucleus and cytoplasm,interacting with related transcription factors to regulate transcription.In recent years,ZYX's role in tumors has attracted attention,and it has been reported to play an important role in the proliferation,migration,invasion and metastasis of tumors such as melanoma,liver cancer,oral squamous cell carcinoma,Ewing's sarcoma,and prostate cancer.However,the role of ZYX in gastric cancer has not been reported.This study examined the expression of ZYX in gastric cancer tissues,and analyzed the relationship between the level of ZYX expression and clinical pathological parameters and clinical prognosis.The roles of ZYX in promoting the invasion and metastasis of GC cells were investigated and the underlying molecular mechanisms were explored.In addition,we also investigated the role of ZYX in glioma migration and invasion.The main methods,results and conclusions of this study are as follows:1.Expression of ZYX in gastric cancer and its clinicopathological significance(1)ZYX is highly expressed in gastric cancer tissuesImmunohistochemical staining was used to detect the expression of ZYX in tissue microarray of 208 cases of gastric cancer and 17 cases of paracancerous tissues.The results showed that the expression of ZYX in gastric cancer tissues was significantly higher than that in paracancerous tissues(p < 0.0001).To confirm these results,Real-time quantitative PCR(qRT-PCR)and Western blot(WB)were used to detect the expression of ZYX in 6 pairs of fresh gastric cancer tissues and adjacent tissues.The results showed that ZYX expression was significant higher in gastric cancer tissues than that in adjacent tissues at both mRNA and protein levels.The analysis results of TCGA database data(p <0.05)also support our experimental results.(2)The expression level of ZYX in gastric cancer tissues is positively correlated with clinical pathological parameters and negatively correlated with patients' prognosisA cutoff value of 6.5 for IHC scores was determined by drawing the ROC curve of the ZYX IHC scores of 208 GC tissues with SPSS20.0.According to the cutoff value,gastric cancer patients were divided into ZYX high expression group and low expression group.Then the correlations between the expression of ZYX and the clinicopathological parameters of GC patients were analyzed.The results showed that the levels of ZYX expression were significantly correlated with tumor size(p = 0.015),T stage(p = 0.002),N stage(p = 0.001)and TNM classification(p = 0.026).Kaplan�Meier survival analysis showed that GC patients with high expression of ZYX had shorter overall survival(OS)(p <0.0001)and progression free survival(PFS)(p <0.0001)compared to the cases with low expression of ZYX.KMPLOT data analysis corroborated the results from our cohort.Univariate analysis of variance and multivariate analysis of variance showed that ZYX can be used as an independent prognostic indicator of gastric cancer.2.ZYX enhances the capabilities of gastric cancer cells to migrate,invade and metastasize(1)Construction of ZYX-overexpressed XN0422 cells and ZYX-knockdown MGC803 cellsThe expressionlevels of ZYX in gastric mucosal epithelial cells(GES-1)and several gastric cancer cell lines BGC823,MGC803,SGC7901,and XN0422(a primary gastric cancer cell line kept in our lab)were screened byusing qRT-PCR and WB.The results showed that the expression of ZYX in each gastric cancer cell line was significantly higher than that in normal gastric mucosal epithelial cells.Among gastric cancer cell lines,MGC803 had the highest ZYX expression level,while XN0422 had the lowerest expression level.Therefore,lentiviral methods were used to construct ZYX-knockdown and ZYX-overexpression models,respectively.After that their efficiencies were verified by qRT-PCR and WB,and they were used in follow-up experiments.(2)ZYX promotes the migration and invasion of GC cells in vitroThe wound healing assay results showed that compared to control cells,overexpression of ZYX significantly enhanced the migration ability of XN0422(p = 0.0001),and knocking-down ZYX significantly reduced the migration ability of MGC803(p < 0.0001).Consistent with the migration experiment results,the Metrigel-transwell invasion experiments also showed that compared to control cells,overexpression of ZYX significantly enhanced the invasion ability of XN0422(p <0.0001),while knocking-down ZYX significantly reduced the invasion ability of MGC803(p < 0.0001).(3)ZYX promotes metastatic capacity of gastric cancer cells in vivoThe mouse abdominal cavity metastasis model showed that XN0422 cells overexpressing ZYX formed significantly more abdominal cavity metastases than control cells(p=0.0003),while MGC803 cells that knocked down ZYX formed significantly fewer abdominal metastases than control cells(p <0.0001).3.ZYX activates WNK1 / Snail pathway to induce EMT in gastric cancer cells(1)Phosphokinase antibody chip screening of ZYX downstream phosphokinasePhosphokinase antibody chip analysis of ZYX's downstream phosphokinases revealed that overexpression of ZYX resulted in up-regulation of eight phosphokinase phosphorylation levels in XN0422 cells,with WNK1 at top,suggesting that WNK1 may be the main downstream phosphokinase molecule regulated by ZYX.This result was verified by WB experiments in XN0422 cells overexpressing ZYX and MGC803 cells knocking-down ZYX.(2)Knockdown of WNK1 and treatment with WNK1 inhibitors significantly inhibited the role of ZYX promoting the invasion of GC cellsAfter silencing WNK1 with small interfering RNA(siRNA)or treatment with WNK1 inhibitor(WNK463)in ZYX-overexpressing XN0422 cells,the in vitro invasion ability of the cells was significantly reduced as compared with the control group.(3)ZYX activates WNK1 / Snail signaling pathway to induce gastric cancer EMTOverexpression of ZYX resulted in up-regulated expression of Snail and N-Cadherin and down-regulated expression of E-Cadherin in XN0422 cells,while knockdown of ZYX led to down-regulated expression of Snail and N-Cadherin and up-regulated expression of E-Cadherin in MGC803 cells.The literature has reported that WNK1 can regulate the expression of Snail.So these results suggest that ZYX may induce EMT in GC cells by activating the WNK1 / Snail signaling pathway,thereby promoting the migration,invasion and metastasis of GC cells.in conclusion:1.ZYX is highly expressed in gastric cancer tissues,and its expression level is significantly positively correlated with tumor size,T stage,N stage,TNM stage,and negatively correlated with overall survival and progression-free survival of gastric cancer patients,which is an independent prognosis factor;2.ZYX promotes the ability of gastric cancer cells to migrate,invade and metastasize;3.ZYX promotes the migration,invasion and metastasis of gastric cancer cells by activating the WNK1 / Snail signaling pathway to induce EMT.4.The role of ZYX in promoting the migration and invasion of glioma cells in vitroIn addition,preliminary experiments showed that ZYX could promote the migration and invasion of glioma cells in vitro,laying a foundation for further research on the role of ZYX in gliomas.