| Background:Proliferative vitreoretinopathy(PVR)is a serious complication of retinal detachment and post-operation,which is characterized by the formation of extensive proliferative membrane on the surface of the retina.Retinal pigment epithelium(RPE)is the main cell type in the proliferative membrane.RPE cells migrate to the vitreous cavity and differentiate into myofibroblasts through epithelial-mesenchymal transformation of(EMT).The continuous activation of myofibroblasts leads to the formation of fibrous tissue.Inflammation plays an important role in the initiation of PVR.As an important inflammatory cytokine,TNF-? is involved in the mechanism of many fibrotic diseases.Previous studies have shown that TNF-? acts on RPE cells,induces morphological changes in RPE cells and increases the expression of ?-smooth muscle actin(?-SMA).More and more studies have found that epigenetic mechanisms can promote the development of fibrosis,which involves many important signaling pathways such as transforming growth factor-? / Smads,as well as a variety of epigenetic processes,such as DNA methylation,histone modification,non-coding RNA,etc.,in which we pay more attention to DNA methylation and microRNA(miRNA).DNA methylation is also a target for the treatment of a variety of cancer and immune diseases in recent years.Studies have shown that DNA methylation plays an important role in EMT of RPE cells.MeCP2 is a reader of DNA methylation,which is highly expressed in PVR membrane and transformed RPE cells.MeCP2 participates in EMT,of RPE cells and can promote a variety of tissue and cell fibrosis.Therefore,anti-fibrosis therapy should explore the risk factors related to the initiation of PVR and understand the external and internal relationship.Zebularine is a DNA methyltransferase inhibitor with low cytotoxicity.Micro RNA(miRNA)is a short non-coding RNA,that regulates the stability of target m RNA or protein translation through post-transcriptional mechanisms.Mi RNA-132 has been shown to be involved in inflammatory regulation.It can negatively regulate inflammatory response by enhancing cholinergic anti-inflammatory pathway and inhibit nuclear factor-kappa B(NF-?B)signal pathway.The high expression of miRNA-132-3p inhibits the inflammatory response during acute renal injury and fibrotic transition,indicating that the expression of miRNA-132-3p also has an effect on the fibrosis process of the disease.Mi RNA-132 is an important regulator of the central nervous system,which affects the morphology,memory and cognition of neurons.At present,it has been found that there are many targets,among which the target MeCP2,has been found in the hypothalamus and hippocampus.It can regulate gene transcription by regulating the expression of MeCP2.Referring to the literature,it is found that there are few studies on the effect of TNF-? on the expression of MeCP2 and miRNA-132 in the early stage of PVR.Objective:The purpose of this study was to investigate the effects of different concentrations and treatment time of Zebularine on the proliferation of ARPE-19 cells,the effects of different concentrations of TNF-? on the expression of MeCP2 protein and miRNA-132-3p in ARPE-19 cells in the early stage.Methods:ARPE-19 cells were divided into control group and treatment group.ARPE-19 cells were treated with different concentrations of Zebularine(1,10,50,100,200 ? mol / L)for 24 h,48h and 72 h,respectively.The absorbance at 450 nm was detected by CCK8 method.In addition,ARPE-19 cells were divided into control group,PBS treatment group and TNF-? treatment group.ARPE-19 cells were stimulated with different concentrations of TNF-?(10,20,30,40,50 ng/ml)for 30 min,.The expression of MeCP2 protein was detected by Western Blot.ARPE-19 cells were divided into control group and TNF-? treatment group.ARPE-19 cells were stimulated with different concentrations of TNF-?(10,20,30,40,50 ng/ml)for 24 hours.The expression of miRNA-132-3p was detected by Real-time PCR.The experimental results were statistically analyzed by SPSS23.0 statistical analysis software,and the data were expressed as mean ±standard deviation.In accordance with normal distribution and homogeneity of variance,LSD pairwise comparison or Dunett Test was used to compare the experimental group with the control group,normal distribution was not consistent with the homogeneity of variance,Tamhane's T2 was used to compare or multiple comparisons between groups,and nonparametric test was used to compare the differences between groups.P <0.05 means that the difference is statistically significant.Results:1.The results of CCK8 showed that,compared with the control group,when the treatment time was 24 h,the concentration of Zebularine was 200 ?mol / L can reduce the survival rate of ARPE-19 cells,the difference was statistically significant(P <0.05).When the treatment time was 48 h and 72 h,different concentrations(10,50,100,200 ?mol / L)of Zebularine could reduce ARPE-19 cell survival rate in a dose-dependent manner compared with the control group,the difference was statistically significant(P <0.05).When the concentration of Zebularine was 200 ?mol / L,the inhibitory effect on ARPE-19 cells was the strongest,and the cell survival rate decreased significantly,the difference was statistically significant(P <0.01).2.Western blot results showed that TNF-? had no significant effect on the expression of MeCP2 in ARPE-19 cells,and there was no significant difference between the experimental group and the control group and PBS group(P> 0.05).3.Real-time PCR results showed that when the concentration of TNF-? was 40 ng/m L,miRNA-132-3p,the expression was significantly lower than that in the control group(P = 0.039).There was no significant difference in the concentration of TNF-? at 10,20,30 and 50 ng/m L(p = 0.674,p = 0.347,p = 0.184,p = 0.172).Conclusions:1.Zebularine can inhibit the proliferation of human RPE cells in a dose-and time-dependent manner.It is suggested that methylation plays an important role in the proliferation of RPE cells.2.The early dose change of TNF-? has no obvious effect on the expression of MeCP2 protein in RPE cells.3.TNF-? can decrease the expression of miRNA-132-3p,a negative regulator of inflammatory response in the early stage.Timely and effective anti-tumor necrosis factor therapy plays an important role in delaying the development of PVR,and miRNA-132 is a promising target for the treatment of fibrotic diseases. |
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