Long Non-coding RNA MEG3 Silencing Protects Against Light-induced Retinal Degeneration

Purposes:Excessive illumination may cause retinal photodamage,leading to ocular degenerative diseases such as macular degeneration.As a tumor suppressor gene,lncRNA-MEG3 lacks expression in a variety of tumors and is associated with a variety of neurodegenerative diseases.The mechanism of this study is to explore the mechanism of the protection of retinal photoreceptor cells through Long non-coding RNA MEG3 silencing.Methods:1.The mice were exposed to the 8000LUX intensity white light,and the expression of MEG3 was observed at different time points.The mouse optic rod cells?661W?were exposed to the 2500LUX white light,and the expression of MEG3 at different time points was observed.661W cells were exposed to H₂O₂?100?mol/L?,and the expression of MEG3 at different time points was observed.All of these were detected by qRT-PCR.2.In vivo experiment:661W cells transfected into SCR shRNA,MEG3 shRNA and control group?uninjected?,then exposed to 2500 LUX white light for 24 hours,qRT-PCR detected the changes of MEG3 expression in each group;MTT assays examined cells activity;Calcein-AM/PI staining was used to determine cell apoptosis.3.In vitro experiment::The C57BL/6J mice were injected with Scr shRNA,MEG3 shRNA,and the control group?uninjected?.After 2 weeks of light injury,qRT-PCR was used to detect the changes of MEG3 expression in each group;the electroretinogram?ERG?,Tunel staining,and immunofluorescence?anti-Rhodopsin?were examined respectively;between the function changes and photosensitivity of the photoreceptor cells in each group were observed and the photosensitivity was observed.The apoptosis of the receptor was changed.4.mechanism research:Western blot detects the change of P53 volume by biotinylated MEG3 or antisense MEG3 RNA,and uses p53 antibody in 661W cell extract to carry out RIP experiment;Western blot detect the expression of P53 after light damage 24h.Result:1.With the increase of light exposure time,the expression level of MEG3 in the retina increased significantly;The time and intensity of light and intensity of661W increased,and the expression of MEG3 increased,the difference between the four groups was statistically significant.2.In vivo experiment:The amplitude of A and B waves is obviously reduced by light damage,and MEG3 silencing can reduce the decline of retinal function.Tunel detection shows that the number of retinal cell apoptosis?Tunel positive?increases significantly.After the expression of silent MEG3,the apoptosis of retina cells is significantly reduced;after light injury,the apoptosis of retinal cells is significantly reduced.The out nucleation layer?ONL?was thinner,after the silence of MEG3,the thinning of the outer nucleus and the protective effect of MEG3 on the photoreceptor cells after retinal light injury,and the retina anti-Rhodopsin detection found that the rhodopsin staining signal in the retinal light injury group of the MEG3 silent group decreased obviously,and the MEG3 silence could significantly reduce the staining letter of the rhodopsin.The reduction of the number.3.In vitro experiment:after transfection of 661W cells with MEG3 interfering virus,the expression of MEG3 was significantly reduced;MTT assays showed that MEG3 silencing retained light induced 661 W cell survival.By Hoechst 33342staining andCalcein/PI staining,MEG3 silencing decreased significantly and light induced apoptotic cells were less.4.Mechanism research:MEG3 can interact with MEG3 in the p53-MEG3protein complex;RIP experiment using p53 antibody.q-rtPCR detection shows that p53 exists in the MEG3 gene complex.RNA PULLDOWN and RIP experiments reveal the direct interaction between p53 and proteins.And down regulated the expression of Pro apoptotic protein?Bax?;MEG3 silencing significantly decreased caspase 3/7 activity.Conclusion:the light stimulation of the retina can increase the expression of MEG3 in both in vivo and in vitro,and MEG3 silencing can reduce retinal light damage;MEG3 silence protects 661W cells to induce apoptosis in vitro;MEG3regulates the apoptosis of photoreceptor cells through p53 transcriptional channel.