The Study Of NFAT5 Regulates Adhesion And Invasion In GBM In Vivo And In Vitro

Glioblastoma multiforme(GBM)is the most common malignant brain tumor in the central nervous system(CNS).The high invasive growth of the tumor contributes to poor prognosis to GBM patients,and the median survival time is less than 12month.Therefore,it is of great importance to better understand the molecular mechanism of GBM cells invasion and to explore new therapeutic regimen.The balance of sodium ions is an important factor to maintain the normal tissue microenvironment of human body.High sodium plays an important role in tumor invasive growth.Osmotic transcription factor NFAT5 is a key regulator of cell osmotic homeostasis and tumor cell migration and invasion.Studies in a variety of tumors have shown that NFAT5 promotes tumor migration and invasion through the activation of integrin signaling pathway.Integrin-mediated adhesion of(FA)protein complex to extracellular matrix(ECM)plays an important role in glioma invasion to surrounding normal tissues.We aim to study the effect of Na~+/NFAT5 axis on the adhesion and invasion of GBM and its mechanism.Method:Part Ⅰ:(1)A total of 83 specimens of glioma were obtained from Tianjin Huanhu Hospital for immunohistochemical staining and western blot analysis to determine the expression of NFAT5 among the differernt WHO grade of gliomas.(2)Kaplan-Meier analysis was used to analyze the relationship between NFAT5expression and patient prognosis.Part Ⅱ:(1)Western blot was used to detect the effect of Na~+/NFAT5 axis on EMT progression in GBM cells.Scratch test and transwell test were used to detect the effect of Na~+/NFAT5 on the migration and invasion of GBM cells.(2)The function of NFAT5 gene was analyzed by m RNA sequencing,the relationship between NFAT5and GBM cell adhesion was detected by cell-ECM adhesion experiment,the expression of integrin signaling pathway related proteins in NFAT5 overexpressed GBM cells was detected by Western blot,and the effect of NFAT5 on the migration ability of GBM cells in ECM was verified by living cell monitoring experiment.The effect of NFAT5 on the translocation of adhesion plaque in GBM cells was detected by immunofluorescence,and the transcriptional regulation of NFAT5 on integrinβ3was verified by CHIP experiment.Part Ⅲ:The effect of Na~+/NFAT5 axis on the adhesion and invasion of GBM cells was verified by in vivo experiments.Luciferase lentivirus was used to infect glioma cell line U251 to construct U251-NFAT5 cell line and establish in situ model of glioblastoma in mouse brain.The tumor volume of mice was measured regularly by living bioluminescence of small animals.Immunohistochemical staining was used to detect the related protein of EMT and the expression of adhesion-related proteins.Results:(1)The expression level of NFAT5 protein in gliomas was significantly higher than that in normal brain tissues,and the expression of NFAT5 in tumor tissues increased with the increase of WHO grade.Kaplan-Meier analysis showed that the high expression of NFAT5 was related to the short overall survival time of the patients.(2)Western blot showed that Na~+/NFAT5 axis promoted the EMT process of GBM cells;Scratch and transwell test showed that Na~+/NFAT5 axis promoted the migration and invasion of GBM cells;m RNA sequencing analysis showed that NFAT5 gene was functionally enriched in cell adhesion-related signaling pathways;adhesion experiments showed that NFAT5 promoted the adhesion of GBM cells to ECM;Western blot showed that NFAT5 promoted the expression of integrinβ3 and p-FAK(Tyr397)in GBM cells;Living cell monitoring experiments showed that NFAT5 promoted the movement and migration of GBM cells in extracellular matrix;immunofluorescence staining of focal adhesion protein showed that NFAT5 in GBM cells promoted the maturation and translocation of cellular adhesion plaques;CHIP experiments showed that NFAT5 had transcriptional regulation on integrinβ3.(3)In the in situ animal model of GBM,Na~+/NFAT5 promoted adhesion and invasive growth of GBM cells in mice.Conclusion:(1)The expression of NFAT5 is increased in gliomas and is closely related to the poor prognosis of patients;(2)NFAT5 promote translocation of adhesion plaques by regulating integrin signal pathway and promote adhesion and invasion of GBM cells;(3)The experimental results in vivo and in vitro are consistent.