| Objective:To explore the role of exocrine derived from human umbilical cord mesenchymal stem cells in the malignant transformation of mouse colitis induced by azomethane oxide and sodium dextran sulfate,and to clarify the role and mechanism of SUMO1-mediated SUMOylation in the inhibition of malignant transformation of mouse colitis by hucMSC-Ex.Methods:1)Isolation and identification of hucMSC-Ex: Isolate human umbilical cord mesenchymal stem cells,extract hucMSC-Ex from the hucMSC culture supernatant,and use NanoSight,transmission electron microscopy,and western blot techniques for identification.2)Randomly divide approximately 20 g of male BLBL/C mice about 6 weeks into 3 groups,AOM/DSS group,and hucMSC-Ex group.Except for the Normal group,the other two groups were induced with AOM/DSS colorectal cancer model in mice.The hucMSC-Ex group was treated with hucMSC-Ex tail vein injection on the 10 th,24th and 38 th day of modelling and on the 70 th day all mice were sacrificed.Observe the body weight,stool characteristics,and colorectal view of mice,compare the tumor formation rate and number of colorectal tumors in each group,and observe the histopathological scores by H&E staining,immunohistochemical detection of SUMO1 expression in tissues,real-time fluorescence quantitative polymerase chain Response analysis of the expression of inflammatory factors(IL-1?,IL-6,IL-10,TNF-?)and the expression level of SUMO1 and its related molecules(SENP1,UBC9)in colorectal tissues,and immunoblot detection in colorectal tissues The expression levels of SUMO1 and its related molecules(SENP1,UBC9,?-catenin)were used to comprehensively evaluate the effect of hucMSC-Ex on AOM/DSS-induced malignant transformation of colitis in mice.3)Human colorectal cells(FHC)were cultured in vitro,and the inflammatory environment was simulated by lipopolysaccharide.HucMSC-Ex was added to relieve inflammation.The cells were divided into three groups: NC,LPS,and hucMSC-Ex groups.After 12 h of co-cultivation,the cells were collected to extract cellular proteins and total RNA.Detect the expression of SUMO1 and its related molecules in cells by immunofluorescence analysis,analyze the expression level of SUMO1 and its related molecules in cells by QRT-PCR,detect the expression of SUMO1 and its related molecules in cells by western blot,and detect SUMO1 and ?-catenin by IP binding situation.4)Compared with normal group mice,the expression of SUMO1 and ?-catenin in colorectal tissue of AOM/DSS group increased,while the expression level of SUMO1 and ?-catenin in colorectal tissue of hucMSC-Ex group decreased,and the Binding is inhibited.In vitro simulation of cell inflammation environment,FHC cells stimulated by LPS,compared with the NC group,the expression level of SUMO1 in the cells of the LPS group increased,the combination of SUMO1 and ?-catenin increased,after treatment with hucMSC-Ex,the expression level of SUMO1 in cells Reduced,the binding of SUMO1 to ?-catenin is inhibited.5)The database is compared with the hucMSC-Ex miRNA sequencing results to screen the key molecules of miRNA.HucMSC-Ex treated cells found that miR-146 a increased in expression level,and determined that it was a key molecule.Transfection of FHC cells showed that miR-146 a mimics inhibited SUMO1 expression most obviously.Results:1)The hucMSC-Ex was successfully isolated,about 100 nm,and the surface markers expressed CD63,CD9 and CD81.2)AOM/DSS-induced colorectal cancer model in mice was successfully constructed,and obvious colorectal tumors and tumor formation rates were seen.After hucMSC-Ex injection intervention,the colon length,weight change,disease activity index(DAI),and histopathological scores of mice in the hucMSC-Ex group improved compared with the AOM/DSS group.The rates are reduced.3)Comparing the tumor formation of mice at different time points,it was found that hucMSC-Ex inhibited the inflammatory response at an early stage.In the AOM / DSS group,colitis was more severe,the colorectal villi structure was more disordered,pro-inflammatory factors(IL-6,IL-1?,TNF-?)were highly expressed,and anti-inflammatory factors(IL-10)were low-expressed.The hucMSC-Ex group significantly improved colorectal inflammation,significantly down-regulated the expression of pro-inflammatory factors(IL-6,IL-1?,TNF-?)and up-regulated the expression of anti-inflammatory factors(IL-10).4)Compared to the normal group,the expression of SUMO1 in the colorectal tissue of the AOM/DSS group increased,and the expression of ?-catenin also increased.The expression of the SUMO1 in the colorectal tissue of the hucMSC-Ex group decreased.Simulating the inflammatory environment of cells in vitro,compared with NC group,FHC cells were stimulated by LPS,compared with NC group,the expression level of SUMO1 was increased,and the binding of SUMO1 and ?-catenin was enhanced.As a result,the binding of SUMO1 to ?-catenin was inhibited.5)The database was compared with hucMSC-Ex miRNA sequencing results to screen key miRNA molecules.HucMSC-Ex treated cells found that the expression level of miR-146 a increased most obviously,and finally determined that miR-146 a was a key molecule.The transfection of miR-146 a mimics,mimics NC,inhibitor and inhibitor NC into FHC cells showed that miR-146 a mimics inhibited the expression of SUMO1.Conclusion : HucMSC-Ex inhibits AOM/DSS-induced malignant colitis transformation in mice by regulating SUMO1. |
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