Loss Of Usp19 Attenuates Colorectal Carcinogenesis Induced By High Fat Diets In Mice

The incidence rate and mortality of colorectal cancer are the top three in cancer,which seriously threatens human health.Colorectal cancer is a long process,involving Wnt,?-Catenin,K-ras,p53 and DNA mismatch repair proteins.There are p53 mutation,APC gene inactivation and K-ras mutation activation in colorectal cancer,which are also related to the change of gut microorganism.The main risk factors for colorectal cancer include high-fat diet,sedentary lifestyle,obesity,diabetes,inflammatory bowel disease and gut microbial disorders.Gut microorganisms affect a variety of metabolism and signal pathways,and then strongly affect the occurrence and development of colorectal cancer.Gut microorganisms interact directly with the host colonic epithelial cells and influence the progression of colorectal cancer by releasing metabolites.However,the specific molecular mechanisms between high-fat diet,gut microorganisms and colorectal cancer are still poorly understood.In order to explore the relationship between gut microbial imbalance,high-fat diet and colorectal cancer,we first fed mice with normal or high-fat diet for a long time,then treated some mice with antibiotics to eradicate any pre-existing gut bacteria,and killed mice after inducing colorectal cancer with AOM/DSS.It was found that the survival time of mice fed with high fat diets was shorter than that of mice fed with normal diets.Compared with normal or high-fat diet fed mice treated with antibiotics,the number,size and volume of colon tumors in mice fed only high-fat diets were increased significantly.To investigate the relationship between gut microbial disorders and high fat diet in colorectal cancer,the spontaneous model of colorectal and small intestine tumor in Apc^Min/+mice was used.Apc^Min/+mice,which contain APC mutation of germline,are prone to spontaneous multiple intestinal polyposis,as a mouse model of human familial adenomatous polyposis.As in the previous AOM/DSS model,the survival rate of Apc^Min/+mice fed on a high fat diet was significantly reduced,and antibiotics treatment rescued these phenotypes.Apc^Min/+mice fed a high-fat diets were found more and larger tumors in the colorectal than mice fed normal diets,which was not observed in high-fat diet fed mice treated with antibiotics.Similar findings were found in the small intestine of Apc^Min/+mice.This suggests that high-fat diet significantly accelerates spontaneous and AOM/DSS induced colorectal cancer transformation and relies on potential gut bacteria.It is a successful tumor treatment strategy to target proteasome,but the exact role in the pathogenesis of colorectal cancer has not been systematically studied.In order to study these,we systematically screened the high-fat diet induced ubiquitinases,whose expression in colorectal cancer is dependent on gut bacteria.The results showed that in the mice fed with high-fat diet,many transcripts of deubiquitinase,especially the transcripts encoding Usp19,were significantly increased in colorectal cancer,and the effect of antibiotic treatment in colorectal carcinogenesis was significantly weakened.Usp19 protein also increased significantly.In colorectal cancer induced by AOM/DSS,the expression of Usp19 protein increased gradually.Usp19 catalyzes the hydrolysis of ubiquitin by ubiquitin binding protein substrate,which is widely expressed in colorectal and liver tissues.In order to study the effect of Usp19 deletion on colorectal cancer,a Usp19 knockout mouse was constructed with CRISPR/cas9.The results showed that Usp19 deficiency significantly prolonged the survival period of mice fed with high fat diet,and significantly reduced the number and volume of colorectal tumors in mice fed with high fat diet induced by AOM/DSS.Usp19 protein in colorectal cancer of Apc^Min/+mice was also significantly up-regulated.In order to investigate whether Usp19 deletion affects the spontaneous growth of colorectal cancer,we hybridized Usp19 knockout mice with Apc^Min/+mice,and followed up the tumor growth of their offspring.Regardless of diet,Usp19^-/-Apc^Min/+mice survived longer than Apc^Min/+mice,and the burden of colorectal cancer was reduced.Similar results were observed in small intestinal tumors.To further explore the role of Usp19 and gut dysbacteriosis in colorectal cancer,wild-type and Usp19^-/-mice fed a high fat diet were treated with antibiotics in AOM/DSS induced colorectal cancer.Antibiotic treatment significantly prolonged the survival of wild-type mice and reduced tumor load,but had no effect on the above phenotypes of Usp19^-/-mice.This suggests that the high-fat diet mediated intestinal dysbacteriosis promotes colorectal cancer in a Usp19 dependent manner.In order to study the mechanism of action of USP19 protein in colorectal cancer transformation,Flag-USP19 was expressed in HEK293 cells.The protein interacting with ubiquitinase usp19 was identified by immunoprecipitation and mass spectrometry.It was found that ME1 was one of the most obvious proteins interacting with USP19.The interaction between exogenous and endogenous was proved by immunocoprecipitation.It was found that the interaction between the two proteins occurred in UCH domain of USP19 and ME1 amino terminal domain.We will further study whether usp19 mediates the stability of ME1 protein and its effect on the activity of ME1 enzyme.We will further study the specific molecular mechanism of Usp19 promoting colorectal carcinogenesis,which will help to reveal the pathological mechanism of colorectal carcinogenesis induced by high-fat diet,and may provide a target for the treatment of this kind of disease.