Role And Mechanism Of TIPE2 In The Development And Progression Of Colorectal Cancer

BackgroundThe mutualistic relationship between the intestinal microbiota and host involves appropriate immune regulation which is critical for maintaining gut homeostasis.Disorder of this process will lead to intestinal mucosal immunodeficiency,intestinal epithelial injury,intestinal barrier function impairment,ending in intestinal related diseases such as inflammatory bowel disease（IBD）and even colorectal cancer（CRC）.Nevertheless,deregulation of the process results in intestinal inflammation and tumorigenesis through largely unknown mechanisms.TIPE2（tumor necrosis factor-α–induced protein 8-like 2）is preferentially expressed by leukocytes and cells in the intestinal tissues and play an important role in immune homeostasis.Additionally,TIPE2 negatively regulates phagocytosis and oxidative burst of innate immune cells by binding to Rac GTPase.Previous studies have found that TIPE2 deficiency can alleviate intestinal inflammatory response and regulate tumorigenesis in mice.ObjectiveAimed to illustrate the expression of TIPE2 in intestine tissue,and investigate the role and mechanism of TIPE2 in colorectal cancer of mice,which will provide important theoretical basis and new molecular target for the prevention and treatment of colorectal cancer.Methods1.Changes of TIPE2 expression were examined in both chemically induced and spontaneous mouse intestinal tumor models by qPCR and western blot.The protein level of TIPE2 was also examined in human colorectal cancer by western blot.2.Wild type（WT）mice and Tipe2-/-mice were given azoxymethane followed by multiple rounds of dextran sodium sulfate to induce colitis-associated colon cancer （CAC）.Apcmin/+ Tipe2-/-mice were obtained by hybridization of Tipe2-/-mice and Apcmin/+ mice which have been proved suffering spontaneous colorectal cancer during normal feeding.During the model,survival and body weight change rates were recorded.In the end of the model,tumor number and tumor load of colon tissue and distal small intestine tissue were recorded,and other tumor relevant indexes were detected by histology,immunoblots,qPCR.3.Expression of TIPE2 in the colon tissue was detected by flow cytometry for Tipe2gfp/+colon tissue.The bone marrow chimeric mouse model was established,and then CAC was induced by AOM/DSS.In the end of CAC model,tumor number and tumor load of colon tissue were recorded,and the morphological structure of colon tissue was detected by hematoxylin-eosin staining.4.During 14 days of CAC model,body weight change and disease activity index were recorded.At the 14 th Day,length of colon tissue was measured.Additionally,and molecular indices related to inflammation in mouse colon tissue were detected by qPCR and western blotting,and myeloid cell survival in mouse intestinal tissue was detected by flow cytometry.5.Fecal and intestinal mucosa tissues of WT and Tipe2-/-mice were collected at the 14 th day of AOM/DSS model,and the fecal flora was sequenced by 16 S r RNA and further verified by qPCR to explore the changes of fecal and mucosa-related flora.6.The intestinal microbe of WT mice and Tipe2-/-mice were cleared with broadspectrum antibiotics,then CAC was treated with AOM/DSS to collect the corresponding macro phenotypic indicators.In the end,the infiltration of immune cells in the intestinal tissues of mice was detected by flow cytometry.Results1.TIPE2 expression was increased in mouse models of intestinal cancer and human colorectal cancer.In mice with CAC and sporadic intestinal models,TIPE2 deficiency significantly inhibits the occurrence of tumors.2.Through analysis of flow cytometry results,coming up with the conclusion that TIPE2 mainly expressed in CD45+ immune cells.Transplantation of TIPE2-deficient bone marrow into WT mice succeeded to rescue the WT protumorigenic phenotypes,indicating a hematopoietic-specific role for TIPE2.3.TIPE2 deficiency attenuates the severity of CAC by successfully resolve and restrict colonic inflammation and protects colonic myeloid cells from death during colitis.The 16 S r RNA sequencing and qPCR identified significant differences in the composition of microbiota between fecal and mucosal samples from TIPE2-deficient vs control mice.TIPE2 deficiency correlated with decreased numbers of inflammation and tumor-associated bacteria,inflammatory response and intestinal tumorigenesis.4.Administration of antibiotics decreased the occurrence of tumors in WT mice to the levels of those of Tipe2-/-mice.ConclusionsIn studies of mice with vs without TIPE2 protein,TIPE2 promotes intestinal inflammation and tumorigenesis by expanding numbers of microbiota and tumorassociated inflammatory responses.Strategies to block TIPE2-mediated pathway might be developed for colorectal tumorigenesis.