The Role Of CircPVT1 In Retinal Pigment Epithelium Cell Senescence

Age-related macular degeneration（AMD）is a neurodegenerative disease of retinal aging.It is the main cause of vision loss in aging countries.It is mainly manifested by the phagocytosis of the outer disc membrane of optic cells by the retinal pigment epithelium cells.The drusen of the retinal formed,and a series of secondary pathological changes are triggered.Circular RNA is a special kind of non-coding RNA molecule.It forms a closed loop through reverse splicing.Compared with linear RNA,it is less likely to be affected by RNA exonuclease and is more stable.Currently,circular RNA plays an important role in human diseases through mi RNA sponges,especially Tissue aging and age-related diseases.Studies have shown that circPVT1 regulates target m RNA translation by adsorbing micro RNA Let-7 in fibroblasts,thereby affecting cell senescence.In this study,we used hydrogen peroxide to establish a human retinal pigment epithelial APRE-19 cell senescence model,and found that the expression of circPVT1 increased in this model.Furthermore,molecular biological methods are used to study the effect of circPVT1 on the senescence of retinal pigment epithelial cells and its expression pathway.Objective:In the H₂O₂-induced APRE-19 cell senescence model,the relationship between circPVT1 and cell cycle and senescence-related proteins was clarified,and explore the role of circPVT1 in the process of cell aging.Methods:After stimulating cells with different concentrations of hydrogen peroxide,the aging model of ARPE-19 cells was established;cell morphology was observed by light microscope,β-galactosidase staining,and cell growth curve detected by CCK-8 to verify cell aging;flow cytometry Cell analysis technology detects changes in the cell cycle after hydrogen peroxide stimulates senescence;verifies the correlation between circPVT1 and ARPE-19 cell senescence by q PCR;detects the expression of senescence-associated secretory phenotype p21,Lamin B1 and MMP3 by q PCR and Western blot;transfects si RNA After silencing circPVT1,the number of senescent cells was detected byβ-galactosidase staining,the cell cycle was analyzed by flow cytometry after PI staining,and p21,Lamin B1and MMP3 were detected by q PCR and Western blot experiments after silencing circPVT1.Results:The cells were stimulated with different concentrations of hydrogen peroxide for 2 hours,and the number of cells was measured by CCK-8.It was found that the stimulation concentration of hydrogen peroxide was 250μM as the cell’s semi-lethal dose,so 250μM hydrogen peroxide was added to serum-free medium DEME/F12 to treat ARPE-19 cells were used to establish a cell aging model in 2 hours;the Hydrogen peroxide stimulation group was stimulated with 250μM hydrogen peroxide for 2 hours,and the control group was treated with pure serum-free medium.The experimental results showed that the cells in the Hydrogen peroxide stimulation group were large and flat,with vacuolar changes;theβ-galactosidase staining results showed that the senescent cells（blue）in the Hydrogen peroxide stimulation group increased;the CCK-8 results showed that the growth rate of the cells in the Hydrogen peroxide stimulation group slowed down;PI staining results showed that the cells in the Hydrogen peroxide stimulation group had G2 cell arrest;q PCR and Western blot experiments showed that compared with the control group,the expression of circPVT1 in the Hydrogen peroxide stimulation group increased,while the expression of p21 and MMP3 genes and proteins increased,the expression of Lamin B1 gene and protein decreased.Here,the si RNA-NC transfected group is the NC group;the si RNA-NC transfected and given H₂O₂ stimulation is the NC+H₂O₂ group;the si RNA-2 transfected and the H₂O₂ stimulation is given to the SI+H₂O₂ group.The results showed that the percentage of senescent cells（β-galactosidase staining）in the transfected SI+H₂O₂ stimulation group was significantly increased compared with the NC group,the percentage ofβ-galactosidase stained cells in the transfected SI+H₂O₂ stimulation group also increased,but was lower than that in the NC+H₂O₂ group;the results of flow cytometry after transfection showed that compared with the NC group In contrast,the proportion of G2 phase cells in the NC+H₂O₂ group was significantly increased,and G2 phase cell arrest occurred.But after silencing circPVT1,compared with NC+H₂O₂ group,SI+H₂O₂ group had significantly less cell arrest in G2 phase.After transfection,q PCR results showed that compared with NC group,p21 and MMP3 genes in NC+H₂O₂ group The expression increased,and the expression of Lamin B1 gene decreased.Compared with the NC+H₂O₂ group,the expression of p21 and MMP3 genes in the SI+H₂O₂group was reduced.Western blot experiments proved that,compared with the NC group,the expression of p21 protein in the NC+H₂O₂ group increased,and the expression of Lamin B1 protein decreased.Compared with the NC+H₂O₂group,the SI+H₂O₂ group showed a decrease in the expression of p21 and MMP3 genes and proteins,and an increase in the expression of Lamin B1protein.Conclusion:1.Stimulation with 250μM H₂O₂ can induce cell senescence,resulting in a significant slowdown of ARPE-19 cell growth rate and G2 blockade.2.After H₂O₂ stimulates ARPE-19 cell senescence,it can cause changes in the expression levels of p21,MMP3,Lamin B1 genes and protein related to the senescence secretion phenotype.3.The expression of circPVT1 gene increases after H₂O₂ stimulates cells.Silencing circPVT1 can improve cell senescence caused by H₂O₂ and the changes in the expression levels of p21,MMP3,Lamin B1 genes and proteins in the secreted phenotypes of related senescence.