Sequential expression of miR-182 and miR-503 cooperatively targets FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma
Genetic changes in colon cancer are known to parallel the tissue abnormalities associated with the disease, namely adenoma and adenocarcinoma. The role of microRNA dysregulation in dysplastic progression, however, is not well understood. Here, we show that miR‐182 and miR‐503 undergo sequential up‐r...
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| Published in | The Journal of pathology Vol. 234; no. 4; pp. 488 - 501 |
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| Main Authors | , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
Chichester, UK
John Wiley & Sons, Ltd
01.12.2014
Wiley Subscription Services, Inc |
| Subjects | |
| Online Access | Get full text |
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| Summary: | Genetic changes in colon cancer are known to parallel the tissue abnormalities associated with the disease, namely adenoma and adenocarcinoma. The role of microRNA dysregulation in dysplastic progression, however, is not well understood. Here, we show that miR‐182 and miR‐503 undergo sequential up‐regulation and drive the progression of colon adenoma to adenocarcinoma by cooperatively down‐regulating the tumour suppressor FBXW7. We identified that increased expression of miR‐182 is a feature of adenomas. A subsequent increase in miR‐503 expression works cooperatively with miR‐182 to induce transformation of an adenoma to adenocarcinoma. We show that introducing miR‐503 into AAC1 cells, which are derived from a benign adenoma, confers tumourigenic potential. We also demonstrated that blocking both miR‐182 and miR‐503 in HCT116 colon cancer cells resulted in increased FBXW7 expression and significantly reduced tumour size in xenograft models. We confirmed relevance of these results in patients by examining the expression levels of miR‐182 and miR‐503 in over 200 colon cancer patients with 12 year survival outcome data. Decreased patient survival was correlated with elevated expression of both miRNAs, suggesting that elevated levels of both miR‐182 and miR‐503 define a novel prognostic biomarker for colon cancer patients. In conclusion, we show that a sequential expression of miR‐182 and miR‐503 in benign adenoma cooperatively regulates the tumour suppressor FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma and miR‐182 and miR‐503 may prove to be novel therapeutic targets. Array data are available at: http://www.oncomir.umn.edu/ Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd |
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| Bibliography: | Colon Cancer Surgical Oncology Training Programme Department of Surgery, University of Minnesota AppendixS1. Supplementary Materials and MethodsFigure S1. Transfection efficiencies of cell lines. (A) HCT116 transfected with miRzip503 with 80% efficiency. (B) DLD1 transfected MZ182, with ˜80% efficiency. (C) AAC1 transfected with miRzip182 by Neon, transfection with > 80% efficiency.Figure S2. Representative picture of puromycin selection. Stable expression of miRzip-182 in HCT116 cells by puromycin selection. Light (A) and fluorescence (B) microscopy of HCT116 cells transfected with miRzip-182 on day 28 after transfection, day 23 after puromycin selection. Light (C) and fluorescence (D) microscopy of HCT116 cells stably expressing miRzip-182, 2 months after selection; the pictures were taken 10 days after withdrawing puromycin.Figure S3. AAC1 cells stably expressed TripZ-miR-503 vector after puromycin selection. (A) Tet-ON-induced miR-503 over-expression in the AAC1 cells stably transfected with Tripz503; data are mean ± SD; *p < 0.05. Light (B) and fluorescence (C) microscopy of AAC1 cells with TripZ-503 under Tet-ON induction; pictures were taken on day 11 after transfection, day 9 after puromycin selection. Light (D) and fluorescence (E) microscopy of AAC1 cells with TipZ-503 under Tet-ON induction; pictures were taken on day 31 after transfection, day 29 after puromycin selection.Figure S4. Control stains for LNA in situ hybridization (LNA-ISH) in human colon cancer tissue sections. (A) LNA-ISH staining with DIG-labelled U6 probe detected high expression of U6 RNA (shown in dark blue); (B) LNA-ISH staining with DIG-labelled scrambled miRNA control probe shows no specific staining.Figure S5. Controls stains for immunohistochemistry (IHC) in human colon cancer tissue sections. (A) Low non-specific staining in human colon cancer section processed with the same IHC protocol, except for with absence of primary antibody incubation; (B) and (C) IHC staining on the sections from the same colon cancer samples with β-actin antibody; both well differentiated (WD) and poorly differentiated (PD) regions show high expression of the housekeeping gene β-actin (shown in brown).Figure S6. FBXW7 protein levels are rescued by blocking miR-182 and/or −503 with miRzip-182 (MZ182) or miRzip-503 (MZ503) in DLD1 colon adenocarcinoma cells.Figure S7. Western blot showing inhibition of miR-182 increased expression of FBXW7 in AAC1 adenoma cells relative to control vector transfection.Figure S8. (A) ECIS assay monitoring proliferation of DLD1. Blocking miR-503, alone or together with miR-182, significantly slows down cell proliferation in DLD1 cells. (B) Blocking miR-182 and/or miR-503 induced cell death in DLD1 colon carcinoma cells; the experiments were repeated three times.Figure S9. ECIS assay monitoring proliferation of HCT116 FBXW7−/− cells. Blocking miR-503 and/or −182 did not alter cell proliferation.Table S1. Primers used in this study.Table S2. Outcomes data for colon cancer patient samples. Department of Surgery laboratory istex:452333227A93727E50EFF166C0C2FC2478E1CC63 ArticleID:PATH4407 ark:/67375/WNG-1NR961WR-C ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0022-3417 1096-9896 |
| DOI: | 10.1002/path.4407 |