Repression of MicroRNA Function Mediates Inflammation-associated Colon Tumorigenesis.
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.



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.



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.



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.
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Takeshi
T
Yoshikawa
State Key Laboratory of Cellular Stress Biology and School of Life Sciences, Xiamen University, Xiamen, China.
Jianfeng
J
Wu
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Japan Science and Technology Agency, PRESTO, Kawaguchi, Saitama, Japan. Electronic address: otsukamo-tky@umin.ac.jp.
Motoyuki
M
Otsuka
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Takahiro
T
Kishikawa
Division of Gastroenterology, The Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan.
Nobumi
N
Suzuki
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Akemi
A
Takata
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Motoko
M
Ohno
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Rei
R
Ishibashi
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Mari
M
Yamagami
Division of Advanced Genome Medicine, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Ryo
R
Nakagawa
Division of Advanced Genome Medicine, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.
Naoya
N
Kato
Department of Immunology, Faculty of Medicine, Kindai University, Osaka-Sayama, Osaka, Japan.
Masaaki
M
Miyazawa
State Key Laboratory of Cellular Stress Biology and School of Life Sciences, Xiamen University, Xiamen, China. Electronic address: jhan@xmu.edu.cn.
Jiahuai
J
Han
Department of Gastroenterology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Kazuhiko
K
Koike
D000818:Animals; D001397:Azoxymethane / Q000633:toxicity; D018938:Caco-2 Cells; D063646:Carcinogenesis / Q000187:drug effects* / Q000235:genetics; D002277:Carcinoma / Q000139:chemically induced / Q000235:genetics*; D045744:Cell Line, Tumor; D003092:Colitis / Q000139:chemically induced / Q000378:metabolism*; D003106:Colon / Q000187:drug effects* / Q000378:metabolism; D003110:Colonic Neoplasms / Q000139:chemically induced / Q000235:genetics*; D003564:Cytidine Deaminase / Q000235:genetics; D016207:Cytokines / Q000494:pharmacology*; D053487:DEAD-box RNA Helicases / Q000235:genetics; D016264:Dextran Sulfate / Q000633:toxicity; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007249:Inflammation; D053582:Interleukin-1alpha / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology; D051379:Mice; D035683:MicroRNAs / Q000187:drug effects* / Q000235:genetics; D043244:Ribonuclease III / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D016207:Cytokines; D053582:Interleukin-1alpha; D053583:Interleukin-1beta; C531907:MIRN122 microRNA, human; C532045:MIRN29a microRNA, human; D035683:MicroRNAs; D014409:Tumor Necrosis Factor-alpha; C487931:mirnlet7 microRNA, human; D016264:Dextran Sulfate; C509781:Dicer1 protein, mouse; D043244:Ribonuclease III; C503504:Apobec3 protein, mouse; D003564:Cytidine Deaminase; D053487:DEAD-box RNA Helicases; D001397:Azoxymethane
Gene Regulation; IBD; Mouse Model; Post-Transcriptional Regulation