Anti-apoptosis Effect Of EZH2 On Renal Tubular Epithelial Cells And Its Mechanism

BackgroundEpigenetic modifications plays an important role under physiological and pathological conditions,affecting the expression of genes involved in cell cycle regulation,energy metabolism,cell proliferation and so on.There are three main forms of epigenetic modification:DNA methylation,genomic imprinting and histone modification.In the histone modification,the most studied is histone methylation modification and acetylation modification.The enhancer of zeste homolog 2(EZH2)is a catalytic subunit of the Polycomb repressive complex 2(PRC2)which contains multiple proteins for its diversified functions,and induces the trimethylation of the histone H3 lysine 27(H3K27me3),acting as a transcriptional repressor.EZH2 over expression is detected in many kinds of tumors,such as breast cancer,colorectal cancer,prostate cancer.EZH2 can inhibit the expression of several key tumor-suppressor genes by its histone methylation function,and accelerate the poor prognosis of tumors.According to these studies,EZH2 has been regarded as a reliable biomarker for tumor progression and can serve as a potential parameter in malignant degree and poor predition of outcome.Despite its crucial role in turmer,EZH2 also has been proved to have a significant impact in non neoplastic disease,for instance,dental pulp inflammation and prostatitis.However,researches about the role of EZH2 in kidney disease are not much,even so,the evaluation of its function in kidney disease is controversial in the academic field.According to the above analysis,we raise a query that what kind of role dose EZH2 play in normal renal tubular epithelial cells?ObjectiveTo investigate the effect of decreased expression of EZH2 on the survival state of NRK.52E cells and the underlying mechanisms.MethodsStimulating NRK.52E cells with different concentrations(10μM,20μM,40μM)of 3-DZNep for 24 hours,or with 20μM 3-DZNep stimulated NRK-52E cells for different time(12 hours,24 hours,36 hours,48 hours).Then collect cells,detecting cell apoptosis rate with flow cytometry,or testing the protein level of EZH2,Deptor,Bax,Bel-2,HuR,Akt,p～Akt(S473),S6K and p～S6K with immunoblotting method,or exploring the enrichment degree of EZH2 in Deptor promoter region by CHIP assay,to investigate the effect of decreased expression of EZH2 on the survival state of NRK-52E cells.All experiments were performed at least 3 times independently.The statistic alanalysis was performed.by using the SPSS statistical software package SPSS 20.0(SPSS Inc,Chicago,USA).The results are expressed as the mean± SEM,differenees were determined using one-way ANOVA.p values of<0.05 were considered statistically significant.ResultsThe using of EZH2 inhibitor 3-DZNep to reduce the expression level of EZH2 can leading to apoptosis in NRK-52E cells,with the decreasion of H3K27Me3 methylation level in the Deptor promoter regions,the increasion of Deptor transcriptional level,the inhibition of mTORC1 and mTORC2 activity,and the decreasion of HuR expression level,and this can be partly reversed with the silent of Deptor expression.ConclusionThe inhibition of EZH2 can reduce the methylation level of H3K27Me3 in the Deptor promoter regions,increasing the transcriptional level of Deptor,then inhibit the activity of mTORC1 and mTORC2,and cause the decrease of HuR expression level,and eventually induce apoptosis in NRK-52E cells.