Repression of MicroRNA Function Mediates Inflammation-associated Colon Tumorigenesis

Abstract

Background & Aims Little is known about the mechanisms by which chronic inflammation contributes to carcinogenesis, such as the development of colon tumors in patients with inflammatory bowel diseases. Specific microRNA (miRNAs) can function as suppressors or oncogenes, and widespread alterations in miRNA expression have been associated with tumorigenesis. We studied whether alterations in miRNA function contribute to inflammation-associated colon carcinogenesis. Methods We studied the effects of inflammatory cytokines, such as tumor necrosis factor, interleukin-1α (IL1A), and IL1β (IL1B), on miRNA function, measured by activity of reporter constructs containing miRNA-binding sites in their 3′ untranslated regions, in human 293T embryonic kidney, Caco-2, HT29, and HCT116 colon carcinoma cells, as well as dicer+/+ and dicer−/−, and Apobec3+/+ and Apobec3−/− mouse embryonic fibroblasts. Cells were analyzed by immunoblots, immunohistochemistry, and flow cytometry. We generated transgenic mice expressing reporter constructs regulated by LET7B, MIR122, and MIR29b response elements; some mice were given injections of miRNA inhibitors (anti-MIR122 or anti-LET7B), a negative control, or tumor necrosis factor. Liver tissues were collected and analyzed by immunoblotting. Reporter mice were given azoxymethane followed by dextran sulfate sodium to induce colitis and colon tumors; some mice were given the ROCK inhibitor fasudil along with these agents (ROCK inhibitors increase miRNA function). Colon tissues were collected and analyzed by immunohistochemistry, immunoblots, and fluorescence microscopy. Results Incubation of cell lines with inflammatory cytokines reduced the ability of miRNAs to down-regulate expression from reporter constructs; dicer was required for this effect, so these cytokines relieve miRNA-dependent reductions in expression. The cytokines promoted degradation of APOBEC3G, which normally promotes miRNA loading into argonaute 2-related complexes. Mice with colitis had reduced miRNA function, based on increased expression of reporter genes. Administration of fasudil to mice did not reduce the severity of colitis that developed but greatly reduced the numbers of colon tumors formed (mean 2 tumors/colon in mice given fasudil vs 9 tumors/colon in mice given control agent). We made similar observations in IL10-deficient mice. Conclusions We found inflammatory cytokines to reduce the activities of miRNAs. In mice with colitis, activities of miRNAs are reduced; administration of an agent that increases miRNA function prevents colon tumor formation in these mice. This pathway might be targeted to prevent colon carcinogenesis in patients with inflammatory bowel diseases.