The Role Of Vitamin D Receptor (VDR) In Regulating Methyltransferase 14 (METTL14) In The Pathogenesis Of Colitis-related Colon Cance

Part Ⅰ:The incidence rate and risk factors of malignancy in patients with inflammatory bowel disease diagnosed at different agesObjective:To identify and compare the dissimilarities in malignancies and related risk factors between patients with elderly-onset IBD(≥60 years)and adult-onset(18-59 years)IBD in Peking Union Medical college Hospital.Methods:Patients with confirmed IBD,diagnosed at age≥1 8 years,between January 1998 and December 2020 at the Peking Union Medical College Hospital were enrolled.Patients were followed up by telephone,inpatient medical records or outpatient records until June 2021 to obtain clinical characteristics,cancer occurrence and death outcomes.R 4.0.3 was used to calculate the annual incidence rate(IR)of malignant tumors in the cohort of IBD patients with adult-onset and elderly-onset.IBM SPSS 21.0 was used to conduct singlefactor analysis of primary disease characteristics,extra-intestinal manifestations,complications,medication history,etc.Clinical characteristics of these patients were analyzed,and cancer-related risk factors were explored by Cox regression analysis.Results:A total of 1480 patients suffering adult-onset IBD and 129 patients suffering elderly-onset IBD with a median follow-up period of 4.9 years and 4.8 years,respectively,were included.Patients in the elderly-onset IBD group demonstrated an increased overall incidence of cancer than that demonstrated by patients in the adult-onset group(IR 26.9 versus 9.51 per 1000 person-years;relative risk[RR],2.83).Colorectal cancer was the most common malignancy in the two groups,and patients suffering elderly-onset IBD demonstrated a higher incidence of the malignancy(IR,7.07 versus 3.34 per 1000 personyears;RR,2.12).Among the extraintestinal cancers,hematological malignancies(including lymphoid neoplasms and myeloid neoplasms)and urinary tract cancers(including renal and urinary bladder carcinoma)were common in the elderly-onset group(IR,4.24 and 4.24 per 1000 person-years,respectively).whereas thyroid cancer was the most common cancer in the adult-onset group(IR,1.36 per 1000 person-years).Analysis of clinical characteristics revealed that patients with elderly-onset IBD who developed cancer were more likely to have diabetes and urinary lithiasis(15.8%versus 2.6%,P=0.041;78.9%versus 53.2%,P=0.035).In addition,patients in the elderly-onset group had a shorter course from IBD to cancer(4.28 ±4.15 versus 12.1 ± 8.75,P<0.0001).less exposure to immunosuppressants(including azathioprine and thalidomide)(4.28±4.15 versus 12.1±8.75,P<0.0001).rare extraintestinal manifestations(including athralgia and oral ulcer)(0%vs.23.4%,P=0.003;0%vs.24.7%,P=0.002).and higher cancer-related mortality(8.45 versus 0.865,per 1000 person-years;31.5%versus 9.72%,P=0.007).Cox proportional risk regression analysis in the elderly-onset IBD group revealed that diabetes was an independent risk factor for the progression to cancer(hazard ratio[HR],12.53;95%CI[21.379-65.99],P=0.008).Conclusion:The risk of malignancy in patients suffering elderly-onset IBD increased significantly as compared with those with adult-onset group.Therefore,cancer monitoring should be initiated regularly for patients in the elderly-onset group.Part Ⅱ:Expression changes of VDR and m6A related genes in chronic colitis and colitis associated colorectal cancerObjective:To detect the expressions of Vitamin D Receptor(VDR)and N6-methyladenine(m6A)related genes in human and mouse colon tissues with chronic colitis and colitisassociated colorectal cancer,and to explore the relationship between VDR and m6A related genes.Methods:1.Control patients with normal colon(Control,Ctrl,n=6),Ulcerative Colitis(UC,n=6)and Ulcerative colitis associated colorectal cancer(UC-CAC,n=6),treated in Peking Union Medical College Hospital(PUMCH)from 2012 to 2019 were enrolled.And paraffin specimens from these patients were used for immunohistochemistry.2.Wild-type(WT)C57BL/6 mice model administrated with Azoxymethane(AOM)/Dextran Sulfate Sodium(DSS)was established and mice were divided into normal control group(Ctrl,n=6)and AOM/DSS model group(AOM/DSS,n=6).3.Immunohistochemistry(IHC)was to assess the expression of VDR protein in colon tissues from the above patients and mice;Western Blotting(WB)and Quantitive real-time Polymerase chain reaction(qRT-PCR)were used to detect the expression of VDR protein and mRNA in mice tissues.4.The Gene Expression Omnibus(GEO)database was used to screen UC and UC-CAC patients,then R software was used to analyze the mRNA expression differences between UC and UCCAC patients,and the Gene Ontology of the differential genes(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis were completed.The correlation between VDR and m6A genes was analyzed by Spearman correlation method.Results:1.The rate of colon tumor formation in the AOM/DSS model group was 100%(6/6).Compared with the ctrl group,the number of tumors and tumor loads were apparently different(0±0 vs.4.33±3.01,P=0.017;0±0 vs.20.5±16.7,P=0.03).Colon cells in the cancerous area had obvious atypia pathologically,and the predominant cells were medium or highly differentiated adenocarcinoma cells in situ,which confirmed the success of this model.Chronic inflammation was found in the adjacent area.2.Immunohistochemical results showed that VDR protein mostly located in the nucleus of colonic epithelial cells,and there were few stainings in the cytoplasm and membrane.Compared with the ctrl group,the expression of VDR in cancerous area of UC-CAC patients and mice was significantly down-regulated(n=6,5.17±2.00 vs.0.26±0.25.P=0.005;n=3,7.67±0.58 vs.0.83±0.18.P=0.003).WB and qRT-PCR showed that the expression of VDR in the tumor tissues of mice was significantly lower than that of the normal control group(n=3.2.20 ±0.70 vs.1.21±0.24,P<0.05;n=5,0.55±0.21 vs.0.012±0.006,P<0.001).3.Bioinformatics analysis showed that there were 1035 genes differentially expressed between patients with UC(n=43)and UC-CAC(n=10)(P<0.05),among which 1028 were significantly down-regulated and 7 were significantly up-regulated in UC-CAC.Further analysis of m6A-related genes showed that a total of 16 m6A-related genes were down-expressed in UC-CAC.Correlation analysis of VDR and m6A gene in UC-CAC showed that methyltransferase-likel4(METTL14)was significantly positively correlated with VDR(r=0.51,P<0.05).Conclusion:The expression of VDR and m6A-related genes were decreased in colitis associated colon cancer tissues,and there is a positive correlation between VDR and m6Aencoding gene METTL14.Part Ⅲ:The mechanism of VDR regulating METTL14 in the pathogenesis of Colitis-associated colorectal cancerObjective:To investigate the relationship between VDR and METTL14 and its role in the pathogenesis of colitis-associated colorectal cancer.Methods:1.The AOM/DSS animal model was established in VDR-konck-out(KO)mice.Low-dose of AOM/DSS(7.5mg/kg AOM+3 rounds 1.5%DSS)was used for the animal model.Mice were separated into 3 groups:VDR-KO control group(KO-Ctrl.n=3).wildtype mice group with low-dose AOM/DSS(WT-LAOM/DSS,n=12)and VDR-KO mice model group(KO-LAOM/DSS,n=7).2.METTL14 mRNA and protein expression was detected in mice by qRT-PCR and Western blot.3.Lentivirus packaging method was used to construct VDR overexpression/knockdown cell lines in colon cancer cell lines DLD-1,HCT-116,SW620 and Caco2.The expression of METTL14 protein and mRNA were detected by WB and qRT-PCR.Immunofluorescence(IF)and Co-Immunoprecipitation(Co-IP)were used to observe the spatial localization of METTL14 protein and VDR protein in SW620,DLD-1 and HCT-116.Finally,the binding of transcription factor VDR to the METTL14 promoter region was quantitatively analyzed by Chromatin Immunoprecipitation(ChIP)and qPCR.Results:1.Compared with WT-LAOM/DSS group,VDR-KO mice in KO-LAOM/DSS group had higher mortality(0%vs.28.6%),more significant weight loss(P=0.038),and higher tumor formation rate(0%vs.100%).The number of tumors was also significantly higher in KO-LAOM/DSS group(0±0 vs.2.60± 1.52,P=0.0001).2.Compared with the normal colon tissue of wild-type mice,the mRNA and protein expression of METTL14 in the normal colon tissue of VDR-KO mice were significantly decreased(n=5,0.032±0.013 vs.0.0086±0.0021,P=0.013;n=3,0.48±0.12 vs.0.22±0.09,P=0.043).Compared with WT-LAOM/DSS/DSS group and colon tissue adjacent to tumor in VDR-KO,METTL14 protein expression was further decreased in VDR-KO tumor tissue(n=3,1.03±0.03 vs.0.19±0.03,P<0.001;n=3,0.53±0.16 vs.0.19±0.03,P=0.046).3.Compared with the control group.METTL14 expression was significantly up-regulated after VDR overexpression in colon cancer cell lines DLD-1.HCT-116 and SW620(n=3,0.26±0.02 vs.0.34±0.03,P=0.016;0.19±0.03 vs.0.67±0.04,P<0.001;0.15±0.02 vs.0.36±0.042,P=0.001);METTL14 expression was significantly down-regulated in colon cancer cell lines DLD-1 and Caco2 after VDR knockdown(n=3.0.75±0.01 vs.0.30±0.09,P=0.001:0.92±0.09 vs.0.45±0.12,P=0.005).4.Immunofluorescence assay showed that VDR and METTL14 co-located in the nucleus,and further co-IP assay confirmed the interaction between VDR and METTL14.Prediction analysis of METTL14 promoter region showed that VDR had two potential binding sites,then we used ChIP-qPCR further proved that VDR was able to bind to the METTL14 promoter region and participate in the regulation of METTL14 expression.Conclusion:VDR plays an important role in preventing the transformation of chronic colitis-associated colorectal cancer,partly by affecting the expression of METTL 14.