Exploration Of The Mechanism Of Stromal Gla Protein In Colorectal Carcinogenesis Of Ulcerative Colitis

Part Ⅰ Clinical feature analysis of ulcerative colitis associated colorectal cancerObjective:To analyze the clinical features of ulcerative colitis associated colorectal cancer(UC-CAC)according to the data of inpatient in Peking Union Medical Hospital.Methods:869 inpatients with Ulcerative Colitis(UC)in Peking Union Medical Hospital from January 1998 to January 2018 were continuously enrolled.Clinical data and the outcome of colorectal cancer were collected via medical records and telephone follow-up.Chi-square test and logistic regression model were used to analyze the data by SAS 9.4.Results:16 patients in 869 UC inpatients were diagnosed CRC during a period of 7548 person years and the incidence rate of UC-CAC was 1.84%.Compared to UC inpatients without CAC,a longer course of disease(OR 1.087,95%CI 1.046-1.129),a lower usage rate of 5-ASA(OR 0.218,95%CI 0.052-0.915)and a higher incidence rate of intestinal stenosis(OR 16.533,95%CI 3.824-71.478)were found in UC inpatients with CAC.Conclusions:A long disease course is a risk factor for UC patients developing CAC,while 5-ASA therapy can reduce the risk of suffering from CAC.For UC patients with intestinal stenosis,CAC should be warned for occurring.Part Ⅱ Expressions of Matrix Gla Protein and some related proteins in the development of Colitis associated Colorectal cancerObjective:To explore the expressions of Matrix Gla Protein(MGP)and some related proteins in the development of Colitis associated Colorectal cancer(CAC)according to clinical samples and mice models.Methods:1.8 patients with normal intestinal tissues,7 UC patients and 6 UC-CAC patients were enrolled,location and expression of MGP were assessed by immunohistochemistry(IHC).2.Different mice models were established in C57BL/6 male mice including normal group,chronic colitis group,colon dysplasia group and CAC group.Each group had 5 mice.Control group was given normal saline by intraperitoneal injection following by drinking water.Chronic colitis group was given normal saline by intraperitoneal injection following by three cycles of 2.5%DSS(one cycle of 2.5%DSS was defined as administration of 2.5%DSS for 7 days and drinking water for 14 days).Colon dysplasia group was given 12.5mg/kg AOM by intraperitoneal injection following by two cycles of 2.5%DSS.CAC group was given 12.5mg/kg AOM by intraperitoneal injection following by three cycles of 2.5%DSS.Mice in dysplasia group were killed at the end of 7th week while mice in other groups were killed at the end of 10th week.Pathological evaluations were carried out to confirm success of mice models.Expression of MGP and CD63 mRNA in colon were measured by qRT-PCR,protein expression of MGP,Smad1,pSmad 1/5 were measured by western blot and(or)immunohistochemistry.Results:1.Expression of MGP increased in the colon tissues of UC patients compared with normal control,while the level of MGP in CAC was significantly higher than that in UC patients mainly observed in the cytoplasm and membrane of colon epithelial cells(P<0.05).2.(1)All the mice models including chronic colitis,colon dysplasia and CAC were established successfully.(2)Expressions of MGP mRNA and protein in the colon of CAC were significantly higher than that in the group of chronic colitis and dysplasia.Compared with chronic colitis,colon in dysplasia group tended to have a higher MGP expression,although the difference was not significant.All the group had higher expressions of MGP than that in normal group(P<0.05).(3)Results of western blot showed that phosphorylation of Smad1/5 in colon significantly decreased in CAC compared to control group(P<0.05)and decreased gradually from normal to colitis to dysplasia to CAC.Results of qRT-PCR showed that expression of CD63 mRNA significantly decreased in the colon of CAC group than other groups and decreased gradually from normal to colitis to dysplasia to CAC.Conclusions:In the development of CAC,Expression of MGP mRNA and protein increased and expression of pSmad1/5 protein and CD65 mRNA decreased gradually from normal to colitis to dysplasia to CAC.PART Ⅲ The role of MGP in the Pathogenesis of colitis-associated Colorectal Cancer and its upstream and downstream mechanisms explorationsObjective:To explore the role of MGP in the Pathogenesis of colitis-associated Colorectal Cancer and its upstream and downstream mechanisms.Methods:1.Screening cell lines:the expressions of MGP in human colon cancer cell lines SW620,SW480,HT29,HCT116,CaCO2,DLD1 and normal colon epithelial cells NCM460 and CCD841 were detected by Western Blot.SW480 cell with low expression of MGP was screened and further verified by qRT-PCR.2.Exploration of the function of MGP:(1)After transient transfection of MGP overexpression plasmid into SW480 cells,the proliferation,migration and apoptosis of SW480 cells were detected by thiazolium test(MTT),Transwell assay and flow cytometry.At the same time,the changes of Ecadherin,Vimentin,Slug and Snail of epithelial mesenchymal transformation(EMT)were detected by Western Blot method.(2)After SW480 cells were treated with different concentrations of recombinant MGP(0,1ng/ml,10ng/ml,50ng/ml,100ng/ml,500ng/ml),the proliferation of SW480 cells was detected by thiazolan assay(MTT),and the changes of EMT indexes such as Ecadherin,Vimentin,Slug and Snail were detected by Western Blot method.SW480 cells were stimulated by different concentrations of TNF α(0,10ng/ml,50ng/ml)to detect the expression of MGP protein in inflammatory environment and the effect of recombinant MGP on cell proliferation.3.Exploration of downstream pathway of MGP:(1)48 hours after transient transfection of MGP overexpression plasmid into SW480 cells,RNA was extracted to verify the overexpression of MGP,and then RNAseq analysis was carried out.Ten genes with large upregulation were validated.(2)48 hours after transient transfection of MGP overexpression plasmid into SW480 cells,the changes of downstream Smad1/5 pathway were detected by Western Blot and the expression level of CD63 was detected by qRT-PCR and Western Blot.4.Exploration of upstream pathway of MGP:(1)SW480 cells were treated with different concentrations of vitamin K to detect the expression of MGP,cell proliferation and the level of Smad1/5 phosphorylation.(2)C57BL/6 male mice were used to establish colitis-associated colorectal cancer model with vitamin K intervention.They were divided into the following four groups:normal control group,model control group,30mg/kg/d vitamin K stimulation group,60mg/kg/d vitamin K stimulation group.The CAC modeling method is the same as the second part.The mice in different vitamin K intervention groups were given vitamin K intervention 2 weeks before intraperitoneal injection of AOM,and were given intragastric administration of vitamin K 5 days a week until the end of the model.All the mice were killed at the end of the 12th week.The number and size of colon tumors in different groups were statistically analyzed,and the pathology was evaluated.The expression of Ki-67 in intestinal tissue was detected by IHC,the expression of MGP was detected by IHC,Western Blot and qRT-PCR,the expression of Smad1 and pSmad1/5 was detected by Western Blot,and the expression of CD63 was detected by qRT-PCR.Results:1.(1)SW480 cell had low expression of MGP.(2)After MGP overexpression plasmid into SW480 cells,cell proliferation and cell migration ability decreased,cell apoptosis ability increased,the expression of Ecadherin increased,while the expression of Vimentin,Slug and Snail decreased.(3)After SW480 cells were treated with recombinant MGP,the cell proliferation ability decreased,the expression of Ecadherin increased,and the expression of Vimentin,Slug and Snail decreased.(4)After SW480 cells were stimulated by 10ng/ml and 50ng/ml TNFa,the ability of cell proliferation and the expression of MGP increased,while the ability of cell proliferation decreased after SW480 was treated with recombinant MGP of 500ng/ml and TNFa of 50ng/ml.2.(1)RNAseq results showed that compared with the control group,1912 genes were up-regulated and 1892 genes were down-regulated in MGP overexpression group.KEGG enrichment analysis showed that MGP was involved in Wnt signal pathway,mTOR signal pathway,Hippo signal pathway and so on.In terms of function,MGP was mainly related to cell proliferation,apoptosis,migration,immune inflammation,epigenetics and so on.We verified the ten genes with large up-regulation,and the results were consistent with RNAseq,including the CD63 gene related to cell adhesion.Western Blot result showed that 48 hours after transient transfection of MGP overexpression plasmid into SW480 cells,the level of CD63 protein increased significantly.(2)48 hours after transient transfection of MGP overexpression plasmid into SW480 cells,Western Blot results showed that the phosphorylation of Smad1/5 increased.3.(1)After SW480 cells were treated with vitamin K,the expression of MGP protein increased,the ability of cell proliferation decreased,and the phosphorylation of Smad 1/5 increased.(2)①Compared with the model control group,the number of colon tumors in the vitamin K intervention group had no significant change,but the size of the tumor decreased significantly(P<0.05).There was no significant change in the number and size of tumors in different vitamin K intervention groups(P>0.05).②IHC results showed that the expression of Ki-67 in vitamin K intervention groups were significantly lower than that in model control group,but there was no significant difference between different vitamin K intervention groups(P>0.05).③The results of IHC and Western Blot showed that the expressions of MGP in the vitamin K intervention groups were significantly higher than that in the model and normal control group,and there was no significant difference among different vitamin K intervention groups(P>0.05).The expression of MGP in the model group was significantly lower than that in the normal control group.The results of qRT-PCR showed that there was no significant difference in the expression of MGP among the above groups.④The results of Western Blot showed that the phosphorylation level of Smad 1/5 in the colon tissue of the model control group was lower than that of the normal control group,while the phosphorylation of Smad1/5 in the vitamin K intervention group was higher than that of the model control group.⑤ The results of qRT-PCR showed that the expressions of CD63 mRNA in colon tissues in model control group and 30mg/kg/d vitamin K intervention group were significantly lower than that in normal control group(P<0.05),while the expression level of CD63 mRNA in colon tissue in 60mg/kg/d vitamin K intervention group was higher than that in model control group and 30mg/kg/d vitamin K intervention group,but there was no significant difference(P>0.05).Conclusions:1.MGP can inhibit the occurrence of tumor by inhibiting cell proliferation,promoting cell apoptosis,inhibiting cell migration and the occurrence of EMT.2.Vitamin K can promote the expression of MGP protein,while MGP can activate Smad 1/5 pathway and increase the expression of CD63,which can further inhibit tumor.