The Role Of OGA In The Evolution Of Colitis-Associated Cancer

Background and Objective: Colorectal cancer is the most common malignant tumor in the world.Colitisor inflammatory bowel disease(IBD),as a recurrent chronic inflammation,is one of the high-risk factors for colorectal cancer.Colitis-associated cancer(CAC)is a typical inflammation-related colorectal tumor.In addition to traditional surgery,radiotherapy,chemotherapy and immunotherapy,traditional Chinese medicine has also played an irreplaceable role,but due to the limitations of its research methods,there is a lack of valuable clinical data,which makes it difficu lt to be widely accepted and recognized.The appropriate animal model can maximize the guidance of tumor clinical treatment,for drug sensitivity testing,looking for action targets,so as to carry out accurate treatment.In recent years,studies have show n that abnormal OGlcNAc glycosylation plays an important role in the progress of CAC.At present,there are few studies on the role of OGA in the progression of colorectal cancer,and its role in CAC is still unclear.This study intends to establish an OGA intestinal epithelial specific knockout(OGA-IKO)mouse model,and on the basis of this model,CAC,is induced to discuss the effect of OGA knockout on the occurrence and development of CAC.To provide more reliable basic research for evaluating the mechanism of anti-tumor effect of traditional Chinese medicine.Methods: 1.The OGA intestinal epithelial specific knockout mouse model was established by the principle of Cre/Loxp system.2.Azomethane oxide(AOM)and sodium dextran sulfate(DSS)were used to induce colorectal cancer on the basis of OGA intestinal epithelial specific knockout mouse model,The phenotype was observed and the related indexes of inflammati on and tumor in mice were detected by Western Blot,q RT-PCR and other methods.3.16 S rDNA high-throughput sequencing technique was used to detect the species and abundance of intestinal bacteria in colitis-associated cancer mice,and to analyze the effect of intestinal epithelial OGA on intestinal flora and whether it is involved in the occurrence of colitis-associated cancer.4.The expressions of OGA,OGT and O-GlcNAc in human colon cancer and precancerous lesions were detected by IHC and q RT-PCR methods,and the correlation and progression of colorectal cancer was analyzed.5.Human colon cancer cell lines HCT-116 and RKO were selected to construct OGA knockdown cell line using lentivirus infection method.The effects of OGA knockdown on the proliferation an d metastasis function of colon cancer cells were detected by CCK8 method,plate cloning,cell scratch and Transwell.Results: 1.The successful construction of OGA intestinal epithelial specific knockout mouse model was verified by Western blot and q RT-PCR methods.2.The results of colorectal cancer induction in mice showed that the tumor formation rate of OGA-IKO group was significantly lower than WT group,(IKO(77.8%)vs WT(100%),P < 0.05).The survival rate was significantly increased(IKO(66%)vs WT(92%),P < 0.05).Intestinal epithelial tissue damage was s light,and inflammatory factors IL-6 and IL-10 was decreased.But TNF-? had no significant difference,suggesting that intestinal epithelial specific knockout OGA reduced the occurrence of colitis-associated cancer.3.16 S rDNA sequencing showed that the f lora diversity was incrased,and composition was changed significantly in OGA-IKO induced colon cancer mice.Compared with WT mice,the relative abundance of Bifidobacteriale and Saccharibacteria(anti-inflammatory)in IKO group were significantly increased,the abundance of inflammation-related bacteria such as Enterococcus,Pasteurellales and Lactococcus were decreased,suggesting that may be related to the reduction of intestinal inflammatory after OGA intestinal epithelial specific knockout.4.Compared with paracancerous tissues,the expression of OGA,OGT and O-GlcNAc increased in human colon cancer.The results of OGA expression in precancerous lesions of colon cancer showed that the positive expr ession rates of OGA in adjacent tissue,low grade intraepithelial neoplasia(LGIN),high grade intraepithelial neoplasia(HGIN)and colorectal cancer were 38.5%(5 /13),54.55%(6/11),90%(9/10)and 92.8%(13/14),respectively.The positive expression rates of OGT in adjacent tissue,LGIN,HGIN and CRC were 30.70%(4/13),63.60%(7/11),90%(9/10)and 92.8%(13/14),respectively.The positive expression rates of O-GlcNAc in adjacent tissue,LGIN,HGIN and CRC were 84.60%(10/13),45.4%(5 /11),90%(9/10)and 85.70%(12/14).5.OGA knockdown HCT-116 and RKO colon cancer cell lines were successfully established.OGT compensatly decreased with OGA knockdown,and the O-GlcNAc level increased in cell lines.The results of cell function test showed that OGA knockdown reduced the ability of proliferation and migration of HCT116 and RKO cells.Conclusion: 1.OGA-IKO significantly reduced the occurence of colitis-associated cancer induced by AOM+DSS.2.OGA-IKO may affect the occurrence and development of colitis-associated cancer by altering the composition of intestinal flora.3.OGA,OGT and O-GlcNAc are closely related to the evolution of colorectal cancer.4.OGA knockdown inhibits the proliferation and migration of HCT116 and RKO cells.