Dynamic Change Of DNA Methylase And DNA Demethylase In CAC Mice Colon And The Intervention Of Heat Clearing And Detoxifying Drugs

Objective:Colitis-associated cancer(CAC)is a subtype of colorectal cancer and is closely related to colitis or Inflammatory bowel diseases(IBD).Subtantial date of clinical and basic indicate that inflammation is closely related to cancer and inflammation can promote the occurrence and development of cancer.The recurrent attack of colitis is one of the most important risk factors.Research has confirmed that the changes of DNA methylation can occur in the early stages of cancer.The abnormal condition of DNA methylation is closely related to the occurrence and development of cancer and we can reverse the malignant transformation of cells by correcting the abnormal condition of DNA methylation.Reseaches shou that the potential applications of DNA methylation can be used as a therapeutic taget of colitis related tumor.Based in the research,the subject study probes the relationship between the occurrence and development of Colitis-associated cancer(CAC)and the dynamic changes of DNA methylase/DNA demethylase.The subject study use AOM/DSS model as experimental system to analysis the dynamic change of DNA methylase(DNMT3A?DNMT3B?DNMT1)and DNA demethylase(TET1?TET3?TDG)in the process from colitis to Colitis-associated cancer,and to analysis the pathogenesis of CAC,and to investigate the essentical contact between CAC and DNA methylation,as well as to provide scientific basis for establishing the therapeutic and to explore the intervention mechanism of Traditional Chinese medicine.Contents:1.The impact of heat clearing and detoxifying drugs on DNMT3 Am RNA?TET3m RNA and TDGm RNA of colon tissue of CAC mice.Methods:The CAC models were established by a single abdomen injection of chemical carcinogen azoxymethane(AOM)combined with three cycles free drinking of dextran sulfate sodium salt reagent grade(DSS).Medicated groups were gaveged by corresponding drug and model group equal does of pure water.Collect samples at the end of the fifth week and the eighth week.The expression levels of DNMT3 a,TET3,TDG m RNA in colon of mice were detected by real time polymerase chain reaction(RT-PCR)and the dates were analyzed using statistical analysis.Result:In fifth weeks(pathology:inflammation):Compared to normal control,the expression of DNMT3 Am RNA in model group showed a downward trend(P>0.05),whereas TET3 m RNA,TDGm RNA in model group increased(P<0.05);Compared to model group,the expression of DNMT3 Am RNA in JB group and SASP group increased,whereas TET3 m RNA,TDGm RNA decreased(P<0.01 or P<0.05).In eighth week(pathology:severe dysplasia ang adenocarcinoma formation):Compared to normal control,the expression of DNMT3 Am RNA in model group showed a upward trend(P>0.05),whereas TET3 m RNA,TDGm RNA decreased(P<0.05);Compared to model group,the expression of DNMT3 Am RNA in JB group decreased,whereas TET3 m RNA increased and the expression of TET3 m RNA,TDGm RNA in SASP group increased(P<0.01 or P<0.05).2.The impact of heat clearing and detoxifying drugs on DNMT3A?TET3and TDG protein of colon tissue of CAC mice.Methods:The CAC models were established by chemical carcinogen azoxymethane(AOM)combined with dextran sulfate sodium salt reagent grade(DSS).Chinese medicine Jiu biying+Bai jiangcao and Sulfasalazine is used to intervent in CAC mice.The expression of DNMT3 a,TET3,TDG at protein level by Western blot and the dates are analyzed using statistical analysis.Result:In fifth weeks:Compared to normal control,the expression of DNA methylase DNMT3 A in model group decreased,whereas DNA demethylase TET3,TDG in model group increased(P<0.01 or P<0.05);Compared to model group,the expression of DNMT3 A in JB group increased,whereas TET3 decreased and the expression of DNMT3 A in SASP group increased,whereas TET3,TDG decreased(P<0.01 or P<0.05).In eighth week:Compared to normal control,the expression of DNMT3A?TDG in model group increased,whereas TET3 decreased(P<0.01 or P<0.05);Compared to model group,the expression of DNMT3 A in JB group and SASP group decreased,whereas TET3 increased(P<0.01 or P<0.05).3.The impact of heat clearing and detoxifying drugs on DNMT1?DNMT3B and TET1 protein of colon tissue section of CAC mice.Methods:The CAC models were established by chemical carcinogen azoxymethane(AOM)combined with dextran sulfate sodium salt reagent grade(DSS).Chinese medicine Jiu biying+Bai jiangcao and Sulfasalazine is used to intervent in CAC mice.After molding,neutral buffered formalin fixed colon mucosa,making biopsy,immunohistochemical staining.The semi-quantitative analysis was made by Image Pro Plus.The average optical density values of DNMT3 a,TET3,TDG were calculated and the dates were analyzed using statistical analysis.Result:In fifth weeks:Compared to normal control,the expression of DNA methylase DNMT3 B in model group decreased,whereas DNA demethylase TET1 in model group increased,DNA methylase DNMT1 increased(P<0.01 or P<0.05);Compared to model group,the expression of DNMT1?TET1 in JB group and SASP group decreased(P<0.01 or P<0.05).In eighth week:Compared to normal control,the expression of DNMT1?DNMT3B in model group increased(P<0.05);Compared to model group,the expression of DNMT1?DNMT3B in JB group decreased,DNMT1 in SASP group decreased(P<0.01 or P<0.05).Conclusion:Based on the results of the study,the dynamic change of expression level between DNA methylation(DNMT3A ? DNMT1 ? DNMT3B)and DNA demethylation(TET1?TET3?TDG)in different pathological stage may be related to the transformation from inflammation to colitis-associated cancer.That is,the balance of DNA methylation /DNA demethylation changed,in general it showed that the DNA methylation decreased and the DNA demethylation increased in the stage of inflammation while the DNA methylation increased and the DNA demethylation decreased in the stage of cancer's formation.Both JB and SASP can reverse the change to some extent,thus may intervene the progression from inflammation to colitis-associated cancer.