High Sodium Mediated Phosphorylation Of EZH2 To Activate TonEBP In Glioma

ObjectiveThe abnormal sodium homeostasis is closely related to many aspects of invasive tumors,and the osmotic pressure transcription factor Ton EBP/NFAT5 has been identified as an important regulator of migration and invasion.Therefore,our research group explored the potential impact of Na~+/Ton EBP signal transduction pathway on the occurrence and development of glioma,providing a new diagnosis and treatment direction for the research of glioma.Methods1.Specimens of patients with glioma were collected and Ton EBP expression was analyzed by IHC staining and Western blot to compare the expression of Ton EBP in adjacent tissues and tumor specimens.Kaplan-meier analyzed the relationship between high and low Ton EBP and poor prognosis in patients.2.To explore the effects of Ton EBP and Na ions on the migration and invasion of glioma LN229 and U251 cells.The effects of Ton EBP and Na ions on the invasion ability of glioma LN229 and U251 cells were investigated by 3D gel simulation in vivo.High-throughput m RNA microarray and KEGG analysis of Ton EBP downstream signal pathway.3.Protein molecules interacting with Ton EBP were identified by silver staining mass spectrometry and further verified by co-ip Assay and Proximity Ligation Assay Assay.The effect of Na Cl on the distribution of Ton EBP slurry was verified by co-ip and core-slurry separation experiments.Transcriptional activity of Ton EBP was detected by luciferase and q-pcr.4.The effects of Ton EBP signal transduction in vivo were studied using a mouse in situ model.Immunohistochemistry,immunofluorescence and Western blotting were used to analyze the expression of Ton EBP and related proteins in the tissues of patients with glioma of different grades.Results1.The expression of Ton EBP in gliomas is closely related to the degree of malignancy.The level of Ton EBP in tumor samples was significantly higher than that in normal brain tissues,and the expression of Ton EBP increased with the increase of pathological grade of gliomas.There was a positive correlation between Ton EBP and poor prognosis of gliomas.2.In order to analyze the role of Ton EBP in glioma,EZH2 interacting with Ton EBP was identified by silver staining mass spectrometry;In order to determine whether EZH2 and Ton EBP can work,we found that they can be combined by Co-IP and PLA experiments in cells and tissues;Western blot showed that the nuclear localization of Ton EBP was significantly reduced after EZH2 knockdown;Four Ton EBP target genes were determined by luciferase report test and q-PCR;EZH2knockout significantly reduced the transcription activity of Ton E BP.3.Histone methyltransferase EZH2 is a specific H3K27 methyltransferase.EZH2can act as a transcriptional activator to methylate transcription factors.We used the immunoco-precipitation experiment to find that Ton EBP methylation exists in glioma.Na Cl can enhance the interaction between EZH2 and Ton EBP as well as the methylation of Ton EBP,and the methylation of Ton EBP can be reduced after EZH2 is lowered or the addition of EZH2 inhibitor DZNep.The m RNA expression of Ton EBP target gene was detected by q-pcr and luciferase reporting assay.It was found that S21A reduced the transcriptional activity of Ton EBP.4.Weekly bioluminescence imaging found Sg-Ton EBP could inhibit the growth of tumor in vivo.However,EZH2-WT effectively restored the phenomenon of Sg-Ton EBP induced tumor growth inhibition.5.In the tumor samples of different grades of gliomas,it was found that there was automethylation of Ton EBP by tissue extraction,and the methylation of Ton EBP increased with the increase of clinical grade.Conclusion1.EZH2 interacts with Ton EBP to activate its activity through lysine methylation.2.High sodium activates phosphorylation at EZH2 S21 and mediates Ton EBP methylation.