June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Integral Analysis of Gene Signatures and MicroRNA Expression of Cultured Human Corneal Endothelial Cells in Relation to Their Functions, Cell Senescence, Epithelial-Mesenchymal Transition, and Fibrosis
Author Affiliations & Notes
  • Kazuko Asada
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Munetoyo Toda
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Michio Hagiya
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Kana Nakata
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Morio Ueno
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Naoki Okumura
    Department of Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Noriko Koizumi
    Department of Biomedical Engineering, Doshisha University, Kyotanabe, Japan
  • Junji Hamuro
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Shigeru Kinoshita
    Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan
  • Footnotes
    Commercial Relationships Kazuko Asada, None; Munetoyo Toda, None; Michio Hagiya, JCR Pharmaceuticals Co., Ltd (E); Kana Nakata, None; Morio Ueno, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P); Naoki Okumura, None; Noriko Koizumi, None; Junji Hamuro, None; Shigeru Kinoshita, Senju Pharmaceutical Co (P), Santen Pharmaceutical Co (P), Otsuka Pharmaceutical Co (C), Alcon (R), AMO (R), HOYA (R)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 3227. doi:https://doi.org/
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      Kazuko Asada, Munetoyo Toda, Michio Hagiya, Kana Nakata, Morio Ueno, Naoki Okumura, Noriko Koizumi, Junji Hamuro, Shigeru Kinoshita; Integral Analysis of Gene Signatures and MicroRNA Expression of Cultured Human Corneal Endothelial Cells in Relation to Their Functions, Cell Senescence, Epithelial-Mesenchymal Transition, and Fibrosis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3227. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Premature cell senescence in cultured human corneal endothelial cells (HCECs) is partly due to epithelial-mesenchymal transition (EMT), a phenotypic switch in which HCECs undergo phenotypic changes resulting in dampened cell propagation. In this study, we investigated and compared HCEC gene-signature features between HCECs cultured with or without EMT-like phase transition to understand the molecular aspects underlying the difficulty in propagating HCECs in culture.

Methods: Gene expression signatures of cultured HCECs with no sign of EMT and those with EMT-like phenotypic changes were compared by use of polymerase chain reaction (PCR) array assay. Due to increasing evidence indicating that microRNA (miR) regulates gene functions, we also integrally analyzed miR expression in corneas from distinct donors and cultured HCECs without EMT-like transition.

Results: Fresh donor HCECs elicited completely distinct gene signatures from those of corneal epithelial cells from the same tissues [correlation coefficient (CC), 0.58~0.62]. Of interest, miR profiles were analogous with those from epithelium (CC, 0.95). Irrespective of the presence of EMT-like transition during culture, mRNA-based gene signatures similar to those of fresh tissue were revealed (CC, 0.7~0.8), while those of miR were far distinct (CC, 0.25~0.50). This implicates the crucial roles of miR in regulating the functional genes of HCECs. The expression profile of miR varied dramatically between donors 20 and 60 years of age. In the integral PCR array assay of gene signatures relating to EMT-like transition, fibrosis revealed a significant decrease in the expression of distinct genes. Moreover, many senescence- and cell-cycle-related genes were downregulated in the cultured HCECs with EMT-like transition.

Conclusions: Gene-signature features of HCECs and corneal epithelium cells from fresh donor tissues were quite distinct, yet similar in miR expression. HCEC mRNA expression remained stable during culture, while HCEC miR expression varied widely during culture, thus indicating the fragility of miR expression. EMT-like transition during culture triggered significant changes of miR traits. These findings will hopefully open a new pathway towards efficient in vitro propagation of HCECs.

Keywords: 481 cornea: endothelium • 535 gene microarray  
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