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Upregulation of BMI1-suppressor miRNAs (miR-200c, miR-203) during terminal differentiation of colon epithelial cells

Journal of Gastroenterology volume 57, pages 407–422 (2022)Cite this article

Abstract

Background

MicroRNAs (miRNAs) are key regulators of stem cell functions, including self-renewal and differentiation. In this study, we aimed to identify miRNAs that are upregulated during terminal differentiation in the human colon epithelium, and elucidate their role in the mechanistic control of stem cell properties.

Methods

“Bottom-of-the-crypt” (EPCAM+/CD44+/CD66alow) and “top-of-the-crypt” (EPCAM+/CD44neg/CD66ahigh) epithelial cells from 8 primary colon specimens (6 human, 2 murine) were purified by flow cytometry and analyzed for differential expression of 335 miRNAs. The miRNAs displaying the highest upregulation in “top-of-the-crypt” (terminally differentiated) epithelial cells were tested for positive correlation and association with survival outcomes in a colon cancer RNA-seq database (n = 439 patients). The two miRNAs with the strongest “top-of-the-crypt” expression profile were evaluated for capacity to downregulate self-renewal effectors and inhibit in vitro proliferation of colon cancer cells, in vitro organoid formation by normal colon epithelial cells and in vivo tumorigenicity by patient-derived xenografts (PDX).

Results

Six miRNAs (miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-345) were upregulated in “top-of-the-crypt” cells and positively correlated in expression among colon carcinomas. Overexpression of the three miRNAs with the highest inter-correlation coefficients (miR-200a, miR-200b, miR-200c) associated with improved survival. The top two over-expressed miRNAs (miR-200c, miR-203) cooperated synergistically in suppressing expression of BMI1, a key regulator of self-renewal in stem cell populations, and in inhibiting proliferation, organoid-formation and tumorigenicity of colon epithelial cells.

Conclusion

In the colon epithelium, terminal differentiation associates with the coordinated upregulation of miR-200c and miR-203, which cooperate to suppress BMI1 and disable the expansion capacity of epithelial cells.

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Abbreviations

ASCL2:

Achaete-scute family basic helix-loop-helix (bHLH) transcription factor 2

AXIN2:

Axin 2

BMI1:

B-lymphoma Moloney murine leukemia virus (Mo-MLV) insertion region 1 proto-oncogene, polycomb ring finger

CD44:

Cluster of differentiation 44 antigen

CD66a:

Cluster of differentiation 66a antigen

CEACAM1:

Carcinoembryonic antigen (CEA) cell adhesion molecule 1

CRC:

Colorectal carcinoma

CSC:

Cancer stem cells

EMT:

Epithelial-to-mesenchymal transition

EpCAM:

Epithelial cell adhesion molecule

EPHB2:

Ephrin (EPH) receptor B2

FACS:

Fluorescence activated cell sorting

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

KRT20:

Keratin 20

LGR5:

Leucine-rich repeat-containing G protein-coupled receptor 5

miRNA:

MicroRNA

mRNA:

Messenger RNA

PDX:

Patient-derived xenograft

RNU6B:

U6 small nuclear RNA 6

RT-qPCR:

Reverse transcription quantitative polymerase chain reaction

3D:

Three-dimensional

3ʹUTR:

3ʹ Untranslated region

ZEB1:

Zinc finger E-box binding homeobox factor 1

ZEB2:

Zinc finger E-box binding homeobox factor 2

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Acknowledgements

The authors wish to thank Michael F. Clarke, MD (Institute for Stem Cell Biology and Regenerative Medicine, Stanford University) for his continuing mentorship and invaluable scientific insights. We thank Dr. Seetha V. Srinivasan (Herbert Irving Comprehensive Cancer Center, Columbia University) for editorial assistance during the preparation of this manuscript. We thank Yusuke Akama and Hiromi Yamazaki (TechnoPro Inc., Fujita Health University) and Hiroaki Sakai (Fujita Health University) for exceptional technical assistance. This work was supported by: (1) the Japan Society for the Promotion of Science (JSPS), through a Research Fellowship for Young Scientists (to Shigeo Hisamori), an Overseas Research Fellowship (to Junko Mukohyama) and the Grants-in-Aid for Scientific Research (KAKENHI) program, with specific regard to grants 17K16555 (to J.M.), 15K14381 (to Yohei Shimono), 18K07231 (to Y.S.), 19K09106 (to Y.S.) and 21H02769 (to Y.S.); (2) the Japan-Belgium Research Cooperative Program (to Y.S.); (3) a post-doctoral fellowship from the Uehara Memorial Foundation (to J.M.); (4) a post-doctoral scholarship from The Cell Science Research Foundation (to J.M.); (5) the Empire State Institutional Training Program (DOH01-C30291GG-3450000) of the New York State Stem Cell Science (NYSTEM) agency (to J.M.); (6) a grant from the Princess Takamatsu Cancer Research Fund (to Y.S.); (7) a Grant from Fujita Health University (to Y.S.); (8) an Extramural Collaborative Research Grant from the Cancer Research Institute of Kanazawa University (to Y.S.); (9) a Grant from the Japan Association for Development of Community Medicine (to T.I.); (10) a BD Biosciences 2011 Stem Cell Research Grant (to Piero Dalerba); (11) a 2016 Runyon-Rachleff Innovator Award (DRR-44-16) from the Damon Runyon Cancer Research Foundation (to P.D.); 11) a 2017 Schaefer Research Scholarship from the Vagelos College of Physicians and Surgeons (VP&S) of Columbia University (to P.D.); (12) NIH/NIDDK Grant K08-DK097181 (to Michael E. Rothenberg), NIH/NCI Grant R00-CA151673 (to Debashis Sahoo), NIH/NIGMS Grant R01-GM138385 (to D.S.), NIH/NIDCR Grant R01-DE028961 (to P.D.), and NIH/NINDS Grant R01-NS109858 (to Vincenzo A. Gennarino); (13) the Paul A. Marks Scholar Program of the College of Physicians and Surgeons (VP&S) of Columbia University (to V.A.G.): and (14) the Promotion and Mutual Aid Corporation for Private Schools of Japan (to Y.S.). Research reported in this publication was supported in part through NIH/NCI Cancer Center Support Grant P30-CA013696. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Author notes

  1. Junko Mukohyama

    Present address: Department of Hepato-Biliary-Pancreatic and Gastrointestinal Surgery, International University of Health and Welfare (IUHW), Tokyo, 1088329, Japan

  2. Darius Michael Johnston

    Present address: Department of Molecular and Cellular Physiology, Stanford University, Stanford, CA, 94305, USA

  3. Shigeo Hisamori, Junko Mukohyama, Sanjay Koul, Takanori Hayashi contributed equally to the study and share co-first authorship.

  4. Piero Dalerba and Yohei Shimono contributed equally to the study and share co-last authorship.

Authors and Affiliations

  1. Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, 94305, USA

    Shigeo Hisamori, Michael Evan Rothenberg, Taichi Isobe, Xin Qian, Darius Michael Johnston, Dalong Qian & Yohei Shimono

  2. Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, 6068507, Japan

    Shigeo Hisamori

  3. Department of Pathology and Cell Biology, Columbia University, New York, NY, 10032, USA

    Junko Mukohyama, Sanjay Koul, Luis Enrique Valencia Salazar & Piero Dalerba

  4. Department of Medicine (Division of Digestive and Liver Diseases), Columbia University, New York, NY, 10032, USA

    Junko Mukohyama, Sanjay Koul, Luis Enrique Valencia Salazar & Piero Dalerba

  5. Herbert Irving Comprehensive Cancer Center (HICCC), Columbia University, New York, NY, 10032, USA

    Junko Mukohyama, Sanjay Koul, Luis Enrique Valencia Salazar & Piero Dalerba

  6. Digestive and Liver Disease Research Center (DLDRC), Columbia University, New York, NY, 10032, USA

    Junko Mukohyama, Sanjay Koul, Luis Enrique Valencia Salazar & Piero Dalerba

  7. Columbia Stem Cell Initiative (CSCI), Columbia University, New York, NY, 10032, USA

    Junko Mukohyama, Sanjay Koul, Luis Enrique Valencia Salazar & Piero Dalerba

  8. Division of Gastrointestinal Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, 6500017, Japan

    Junko Mukohyama & Yoshihiro Kakeji

  9. Department of Biological Sciences and Geology, Queensboro Community College (QCC), City University of New York (CUNY), New York, NY, 11364, USA

    Sanjay Koul

  10. Department of Biochemistry, Fujita Health University School of Medicine, Toyoake, Aichi, 4701192, Japan

    Takanori Hayashi, Masao Maeda & Yohei Shimono

  11. Department of Pathology, Fujita Health University School of Medicine, Toyoake, Aichi, 4701192, Japan

    Masao Maeda & Naoya Asai

  12. Genetic Sciences Division (GSD), Thermo Fisher Scientific, South San Francisco, CA, 94080, USA

    Kaiqin Lao

  13. Department of Genetics and Development, Columbia University Irving Medical Center, New York, NY, 10032, USA

    Vincenzo Alessandro Gennarino

  14. Department of Neurology, Columbia University, New York, NY, 10032, USA

    Vincenzo Alessandro Gennarino

  15. Department of Pediatrics, Columbia University, New York, NY, 10032, USA

    Vincenzo Alessandro Gennarino

  16. Initiative for Columbia Ataxia and Tremor (ICAT), Columbia University Irving Medical Center, New York, NY, 10032, USA

    Vincenzo Alessandro Gennarino

  17. Department of Computer Science and Engineering and Department of Pediatrics, University of California San Diego (UCSD), San Diego, CA, 92123, USA

    Debashis Sahoo

Authors
  1. Shigeo Hisamori
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  2. Junko Mukohyama
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  18. Yohei Shimono
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Contributions

Study concept and design: SH, JM, SK, TH, MER, PD, YS. Acquisition of data: SH, JM, SK, TH, MER, TI, MM, XQ, DMJ, DQ, VAG, DS, PD, YS. Analysis and interpretation of data: SH, JM, SK, MER, TI, LEVS, KL, NA, YK, VAG, DS, PD, YS. Drafting of the manuscript: SH, JM, LEVS, PD, YS. Statistical analysis: SH, JM, SK, TH, MM, TI, LEVS, VAG, DS, PD, YS. Obtained funding: SH, JM, PD, YS. Technical or material support: SH, JM, SK, MER, TI, LEVS, DMJ, DQ, KL, YK, PD, YS. Study supervision: PD, YS.

Corresponding authors

Correspondence to Piero Dalerba or Yohei Shimono.

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Conflict of interest

Yohei Shimono is co-inventor on a patent application owned by Stanford University (US-20110021607), describing the use of miRNAs, including miR-200c, as biomarkers for the identification and therapeutic targeting of cancer stem cells. Yohei Shimono holds a financial relationship with a pharmaceutical company that might be considered relevant to this study: Quanticel Pharmaceuticals, now a fully owned subsidiary of Celgene and Bristol Myers Squibb (patent royalties, stock ownership). Piero Dalerba is co-inventor on patents and patent applications owned by the University of Michigan (US-7723112, US-20140030786) and Stanford University (US-9329170, US-09850483) and describing the use of EpCAM, CD44 and CD66a/CEACAM1 as bio-markers for the identification and differential purification of different subsets of colon epithelial cells. Piero Dalerba holds financial relationships with pharmaceutical and biotechnological companies that might be considered relevant to this study, including relationships with: Oncomed Pharmaceuticals, now a fully owned subsidiary of the Mereo BioPharma Group (patent royalties), Quanticel Pharmaceuticals, now a fully owned subsidiary of Celgene and Bristol Myers Squibb (patent royalties, stock ownership), Forty Seven Inc., now a fully owned subsidiary of Gilead Sciences Inc. (patent royalties, stock ownership), Amgen (stock ownership), Alexion Pharmaceuticals Inc. (employment of an immediate family member, stock ownership), AstraZeneca plc (stock ownership), Eli Lilly and Company (stock ownership), Merck & Co Inc. (stock ownership) and Pfizer Inc. (stock ownership). Piero Dalerba received a grant from BD Biosciences. Piero Dalerba received an honorarium from the Samsung Medical Center to give a scientific lecture. Michael E. Rothenberg is co-inventor on a patent application owned by Stanford University (US-20130225435), describing the use of CEACAM1/CD66a as bio-marker for the identification and differential purification of different subsets of colon epithelial cells, and is currently an employee of Genentech, now a fully owned subsidiary of Roche Holding AG. Kaiqin Lao was an employee of Thermo Fisher Scientific, which commercializes some of the reagents used in this study for the analysis of miRNA expression levels, and currently serves as the chief executive officer (CEO) of X Gen US. Shigeo Hisamori, Junko Mukohyama, Taichi Isobe, Luis E. Valencia Salazar, Xin Qian, Darius M. Johnston, Dalong Qian, Yoshihiro Kakeji, Vincenzo A. Gennarino and Debashis Sahoo disclose no conflicts of interest considered relevant to this study.

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Hisamori, S., Mukohyama, J., Koul, S. et al. Upregulation of BMI1-suppressor miRNAs (miR-200c, miR-203) during terminal differentiation of colon epithelial cells. J Gastroenterol 57, 407–422 (2022). https://doi.org/10.1007/s00535-022-01865-9

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Keywords

  • miRNA
  • Colon
  • Cell differentiation
  • BMI1
  • Tumor-suppressor