Experimental Study Of DNA Methylation Modification During Methyl Methanesulfonate Induced Chronic Retinal Damage

Background and objectiveAlkylating agents are a group of cytotoxic drugs for the treatment of malignant tumors.However,Alkylating agents induced DNA toxic damage not only occurs in tumor cells but also affects the healthy tissues.Methyl methanesulfonate(MMS),an S_N2 alkylating agent could specifically induce retinal photoreceptor damage,whereas the other retinal neurons are not sensitive to it.Thus,MMS is wildly used as a kind of chemical mutagens leading to retinal photoreceptor specific injury.However,high dose(75mg/kg)of MMS has obvious side effects on the kidney,brain,pancreas,and other organs,and the damage of retinal photoreceptor cells is acute and serious,which is inconsistent with the chronic retinal toxicity caused by application of chemotherapy drugs.Therefore,it is more practical to establish a chronic drug-induced retinal injured animal model with little systemic toxicity and high specificity for the study of the toxic reaction of chemotherapeutic drugs to retinal photoreceptor cells.Various factors,such as environment,habit,diet and long-term drug treatment,etc.might reprogram the epigenetic modifications,regulate chromatin structure and gene transcription,leading to changes in physiological and pathological processes.MMS,as an exogenous stimulus may also change the epigenetic modifications.However,little is known about how epigenetic mechanisms regulate the process of retinal injury induced by MMS.induce reprogramming of epigenetic modifications,DNA methylation is one of the most stable epigenetic mechanisms,which can be inherited to offspring and plays an important role in the process of many diseases.Studies have confirmed that DNA methylation levels were increased in several retinal diseases,such as retinitis pigmentosa,age-related macular degeneration,and diabetic retinopathy,accompanied by the activation of DNA methyltransferases.It suggests that abnormal DNA methylation may be the initiating factor of neuronal cell death.Inhibiting DNA methylation status may be an effective strategy to protect neurons.Although a large number of studies have proved that DNA hypermethylation is associated with photoreceptor damage in the model of hereditary retinal degeneration,there is no relevant report on whether DNA methylation is also involved in the regulation of drug-induced photoreceptor damage.In this current study,the morphological and functional changes of the retina caused by different doses of MMS were observed,and the most appropriate dose of MMS was selected to establish the chronic drug-induced photoreceptor cell injury model.Furthermore,the changes of DNA methylation profile and DNA methylation related regulatory factors in the MMS injured retina were detected,and the differentially methylated genes which were related to retinal development and disease were screened.Then 5-aza-2’-deoxycytidine(5-aza-dC),an inhibitor of DNA methyltransferase was used to alter the DNA methylation level of the injured retina to explore the regulation and molecular mechanism of DNA methylation in MMS-induced retinal injury.It will provide the experimental and theoretical basis for regulating epigenetic modification as a new therapeutic target to relieve the drug-induced retinal photoreceptor cell injury.Methods:The drug-induced retinal injury model was established by a single intraperitoneal injection of MMS.Various doses of MMS(75,50,37.5,18.75,9.375 mg/kg)were used,and the degree and process of retinal photoreceptor cells damage were observed by ERG,OCT,and retinal pathological section staining.And the most appropriate dose of MMS was chosen to establish the chronic toxic retinal photoreceptor cell damage model.The whole changes in DNA methylation of the mouse retina after MMS administration were estimated by immunofluorescence staining with 5m C antibody.DNA and RNA were extracted from the retinas of mice in the normal control group and the chronic model group,respectively.Using reduced representation bisulfite sequencing(RRBS)and RNA-sequencing to detect the whole genome methylation patterns and gene expression levels of the two groups of retinal tissues.Differentially methylated regions(DMRs)and differentially expressed genes(DEGs)between the two groups were analyzed performed GO analysis and KEGG pathway enrichment via bioinformatics tools.Differential genes related to retinal development,damage,and physiological function were screened,and the m RNA levels were verified by q PCR.Use the western blot technique to detect the levels of DNA methylation associated proteins.Finally,to verify the regulatory effect of DNA methylation on the retina after MMS injury,we used DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine(5-aza-dC)by intravitreal injection.The effect of 5-aza-dC on the visual function and retinal morphology were observed,and the protein levels of DNMTs and methylation differential genes were detected.Results:1.MMS-induced drug toxicity retinal photoreceptor cell damage is time-dose-dependent.The MMS 75mg/kg treatment group showed acute changes.On the 7th day after treatment,the retinal photoreceptor cells were severely damaged,and only 2～3layers of photoreceptor cells in the outer nuclear layer remained.The outer segments disappeared,the retinal function was lost,and the ff ERG reaction Extinguished,the difference is statistically significant compared with the normal control group(P<0.001).The MMS 50mg/kg dose group showed chronic changes.By the 4th week,4 to5 layers of photoreceptor cells in the outer nucleus were left,the outer segment disappeared,and the amplitude of each waveform in the ff ERG dark adaptation 3.0response decreased and the function was impaired,and compared with the normal control group,the difference was statistically significant(P<0.001).2.The HE staining of the liver,kidney,brain and other important organs in the chronic drug-toxic retinal injury model showed no obvious abnormality compared with the normal control group.3.After chronic drug toxicity retinal photoreceptor cells are damaged,the 5m C fluorescence staining of the outer nuclear layer increases,the level of DNA methylation significantly increased,and the expression of DNMT1 and Me CP2 protein increases.RRBS sequencing and RNA-seq correlation analysis found that the DNA methylation level in the promoter region of a transcription factor Crx was increased,while the m RNA expression of which was decreased.The verification results of q PCR were the same.4.5-aza-dC intravitreal injection intervention improved the damage caused by MMS to retinal photoreceptor cells,the thickness of the outer nuclear layer of the retina increased compared with the model group,and the shape of the outer segment recovered.The retinal function has also improved.At the same time,5-aza-dC reversed MMS-induced higher DNMTs expression and lower Crx m RNA levels.Conclusion:1.A single intraperitoneal injection of MMS 50mg/kg can induce specific damage to retinal photoreceptor cells in mice,and successfully establish a chronic drug-toxic retinal photoreceptor cell damage model.The model has good stability,high reproducibility,and has no obvious toxic and side effects on important organs in the body.2.DNA methylation modification is related to the damage of photoreceptor cells induced by MMS,which may be regulated by DNA methyltransferases and Me CP2;and the methylation of Crx,a transcription factor related to retinal development,is involved.3.The DNA methyltransferase inhibitor 5-aza-dC could reduce the expression of DNMT1,then increase the m RNA expression of Crx,and promote the partial recovery of the morphology and function of the photoreceptor cells and the outer segments of the retina.