Research On The Inhibition Of EZH2 Against TGF-β1-Induced Epithelial-Mesenchymal Transition In RPE Cells

Objective: To investigate the anti-epithelial-mesenchymal transition(EMT)effect of inhibiting enhancer of zeste homolog 2(EZH2)in primary human retinal pigment epithelial(RPE)cells and its possible mechanism.Methods:(1)Primary human RPE cells were extracted from the eye tissues of cornea donors,cultured and identified.(2)Transforming growth factor beta 1(TGF-β1)was added to induce EMT in RPE cells.(3)The expression of EZH2 was inhibited by EZH2-siRNA,and the expressions of epithelial and mesenchymal cell markers in RPE cells were detected by q PCR,western blotting and immunofluorescence staining.In addition,the migration ability,cell proliferative ability and barrier functions of RPE cells were observed by cell migration assay,proliferation assay,apoptosis assay,transepithelial electrical resistance assay and the permeability assay.(4)RNA-sequencing(RNA-seq)analysis was performed to screen the downstream targets of EZH2 silencing,and then the key genes in RNAsequence results were verified by q PCR.Results:(1)The result of immunofluorescence staining showed that primary human RPE cells were successfully isolated and cultured.(2)After TGF-β1 treatment,primary human RPE cells acquired the morphology of mesenchymal cells,and the expression of the epithelial marker zonula occluden-1(ZO-1)was reduced,while the expressions of mesenchymal markers type I collagen(collagen1),fibronectin(FN)and α-smooth muscle actin(α-SMA)were up-regulated,which suggested the RPE cells had gone through EMT.(3)The inhibition of EZH2 with siRNA could effectively reduce the levels of collagen1,FN and α-SMA and increase the level of ZO-1.The results of cell migration assay,proliferation assay,apoptosis assay,transepithelial electrical resistance assay and the permeability assay indicated that inhibition of EZH2 could significantly attenuate the migration and proliferative abilities of RPE cells,but didn’t induce cell apoptosis.Meanwhile,EZH2 suppression could enhance their barrier functions.(4)RNA-sequence results showed that the anti-EMT effect of EZH2 inhibition was mainly related to the MAPK signaling pathway,cytokinecytokine receptor interaction and the TGF-β signaling pathway.Conclusions: Inhibition of EZH2 can effectively attenuate the EMT of RPE cells and restore their barrier functions to a certain extent.EZH2 may be a novel therapeutic target against EMT in RPE cells.