The Mechanisms Of DNA Methvlation In The Penetrating Intestinal Mucosal Lesions Of Crohn's Disease.

Crohn's disease is a chronic and recurrent bowel disease that mainly affects the end of the small intestine and the beginning of the colon.Penetrating intestinal mucosal lesions is a common surgical complication of Crohn's disease,and the disease course is rather complicated.However,the study of CD combined with penetrating lesions focused on Crohn's disease combined with perianal penetrating lesions,and less involved in intestinal penetrating lesions.At present,the etiology of Crohn's disease is not yet clear.The mainstream view is that Crohn's disease is caused by complex interactions of multiple factors such as genetic variation,intestinal flora,host immune system,and environmental factors.Epigenetics,as an important fact linking genetic and non-genetic factors,should play an important role in the pathogenesis of Crohn's disease.At the same time,the cause of penetrating intestinal mucosal lesions is not yet clear,the general view is that this is a chronic result of acute inflammation caused by infection or sepsis,and is associated with the formation of deep mucosal ulcers.However,the specific mechanisms and participation factors are not yet clear.DNA methylation,the most widely studied branch of epigenetics,may be able to explain the mechanism of the complex lesions.DNA methylation is catalyzed by DNA methyltransferase,which covalently links a methyl group to the 5th carbon atom of cytosine to form 5-methylcytosine.Early studies focused more on changes in DNA methylation in peripheral blood of Crohn's disease patients,and only a few studies focused on intestinal mucosal tissue of Crohn's disease patients.The intestinal mucosal tissue is the most direct communication site among internal environment,external environment and intestinal flora,and it contains a rich and complex intestinal immune system.Therefore,the DNA methylation state of the Crohn's disease intestinal mucosal tissue is worth studying,and it could provide a new idea for the exploration of mechanism of penetrating intestinal mucosal lesions.PART I Clinical Features of Crohn's Disease Combined with Intestinal Penetrating LesionsOBJECTIVE:This study aimed to analyze the specific clinical features of Crohn's Disease patients with intestinal penetrating lesions by comparing them with those with neither stenosis nor penetrating lesions and stenosis alone.Methods:A total of 343 patients definitely diagnosed with Crohn's disease admitted to the Department of General Surgery,Jinling Hospital,Medical School of Nanjing University from September 2010 to August 2014 were retrospectively collected.According to the Montreal classification criteria,patients were divided into 3 groups:intestinal penetrating lesions(B3 type)group,intestinal stenotic lesions(B2 type)group,and no intestinal penetrating or stenotic lesions(B1 and P types)group.Demographic data and clinical data were compared between groups.RESULTS:The percentage of patients who underwent intestinal surgery in the intestinal penetrating lesions group was significantly higher than that in the intestinal stenotic lesions group(82.2%vs.67.4%,P=0.015)and no intestinal penetrating and stenotic lesions group(82.2%vs.58.1%,P=0.000).Among them,the proportion of patients undergoing ileal resection in the intestinal penetrating lesions group was significantly higher than that in the other two groups(43.3%vs.14.0%,P=0.O00,43.3%vs.9.7%,P=0.019).Penetrating and stenotic lesions.Patients with intestinal penetrating lesions were significantly more likely to receive other small bowel resections(34.4%vs.20.2%,P=0.019)and ileostomys(11.1%vs.1.6%,P=0.005)than those without intestinal penetrating and stenotic lesions.Conclusions:The risk of intestinal surgery was significantly higher in Crohn's disease patients combined with intestinal penetrating lesions compared to other types of Crohn's disease patients.PART II Primary Screening for Differential DNA Methylation Sites in Penetrating Intestinal Mucosal Lesions in Crohn's DiseaseOBJECTIVE:The objective of this study was to use the latest Infinium 850K DNA methylation microarrays to comprehensively assess the methylation changes of whole genome DNA in the intestinal mucosa of adult Crohn's disease patients.Compare DNA methylation status with the healthy people and screen for differential DNA methylation locis.METHODS:Seven patients with Crohn's disease who underwent colonoscopy at Jinling Hospital,Medical School of Nanjing University in November 2016 were randomly selected.With age and sex as the standard,7 healthy people were strictly matched as the control group.In Crohn's disease patients,pieces of intestinal mucosa were taken from the penetrating lesions and the normal parts under endoscope respectively,the mucosa samples were divided into the Crohn's disease penetrating group and Crohn's disease non-penetrating group;the intestinal mucosal samples of healthy people entered the normal group.The Infinium 8 50K DNA methylation chip was used to analyze the methylation abnormalities.RESULTS:A total of 5,200 differential DNA methylation sites were screened in the penetrating intestinal mucosa of Crohn's disease and normal intestinal mucosa.The most diverse hypermethylation sites:KCNJ13;GIGYF2,C7orf72,and HLA-DRB1;demethylation sites:HERPUD2,MUC1,and TMTC2.Comparing penetrating intestinal mucosa with non-penetrating intestinal mucosa of Crohn's disease,a total of 3237 differential DNA methylation sites were screened.The most diverse hypermethylation sites:MTSS1,YPEL5,EFCAB11 and CBLB,and demethylation sites:PLEKHG1,LINC01506 and KIAA0753.Differential DNA methylation sites are involved in the positive regulation of apoptosis and the positive regulation of interleukin-8 production,and are enriched in inflammatory bowel disease signaling pathways and extracellular matrix receptor-receptor signaling pathways.CONCLUSIONS:The differential DNA methylation sites associated with penetrating intestinal mucosal lesions in Crohn's disease include C7orf72,HLA-DRB1,MUC1,YPEL5,and CBLB.Apoptosis,IL-8 production,and abnormal ECM might be involved in the pathogenesis of Crohn's disease penetrating intestinal mucosal lesions.PART III Verification of Differential DNA Methylation Sites in Penetrating Intestinal Mucosal Lesions of Crohn's DiseaseObjective:To verify the results of the Infinium 850K DNA methylation microarrays in previous study by pyrosequencing to reveal the role of related methylation locis in the pathogenesis penetrating intestinal mucosal lesions of Crohn's disease and explore the relationship between DNA methylation and disease activity of Crohn's disease.Methods:Twenty-five Crohn's disease patients who underwent colonoscopy at Jinling Hospital,Medical School of Nanjing University and 7 healthy controls were selected.The patient's age,sex,Crohn's disease activity scores,and other data were collected.Pieces of intestinal mucosa were taken from Crohn's disease patients(penetrating intestinal mucosal lesions)and healthy individuals(normal mucosa).The methylation abnormalities of HLA-DRB1,MUC1,YPEL5,and CBLB locis were detected by pyrosequencing.Results:Compared with the control group,the degrees of methylation of HLA-DRB1,YPEL5 and CBLB locis in Crohn's disease group were significantly increased,while the degree of methylation of MUC1 site was significantly decreased.The relative expression levels of HLA-DRB1,YPEL5,and CBLB in Crohn's disease group were significantly decreased,and the relative expression level of MUC1 was significantly increased.Correlation analysis found that the degrees of methylation of HLA-DRB1(r =-0.62,P<0.001),MUC1(r =-0.45,P = 0.01),YPEL5(r =-0.55,P = 0.001)and CBLB(r =-0.62,P<0.001)was significantly negatively correlated with its relative expression levels.The degree of methylation of MUC1 was significantly negatively correlated with Crohn's disease activity scores(r =-0.50,P = 0.01).Conclusion:The degrees of methylation of HLA-DRB1,YPEL5,and CBLB were markedly elevated in penetrating intestinal mucosal lesions of Crohn's disease,while the degree of methylation of MUC1 was decreased.The degree of methylation of MUC1 was negatively correlated with the disease activity of Crohn's disease.