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Morio Ueno, Kazuko Asada, Munetoyo Toda, Michio Hagiya, Naoki Okumura, Noriko Koizumi, Junji Hamuro, Shigeru Kinoshita; The integral analysis of senescence-associated secretory pathway and microRNA secretion of cultured human corneal endothelial cells relating to their functions, cell senescence, and epithelial-mesenchymal transition. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2076.
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Premature cell senescence in cultured human corneal endothelial cells (HCEC) is partly due to the epithelial-mesenchymal transition (EMT) phenotypic switch, in which HCEC undergo phenotypic changes resulting in damped propagation, partly mediated by the senescence associated secretory pathway (SASP). To elucidate molecular aspects underlying the difficulty in propagating HCEC in culture, features of SASP and microRNA (miR) secretory products were detailed and compared between cell cultures with/without EMT-like phase transition.
Secretory products of distinct cultured HCEC, with no EMT signs, were compared with those with EMT-like phenotypic changes by ELISA, Bio-Plex systems, microarray analysis and PCR array. Considering the relevance of miR in regulating cell-to-cell interaction during phenotypic degeneration, miR expression in culture supernatants of HCEC w/o EMT-like transition was also analyzed.
miR-based gene signatures were far distinct from those of fresh tissues (correlation coefficient: 0.25~0.50) and miR141, 200a, b, c, and 205 were downregulated in HCECs with EMT-like transition, thus indicating the crucial roles of miR in regulating HCEC functional genes during culture. Moreover, the specified species of miRs secreted in the culture medium (e.g., miR1273) were dramatically reduced in HCEC cultures kept in differentiated HCEC phenotype, yet no decrease was found in the culture of HCEC with EMT-like transition. Similarly, secretory products of SASP, IL8, IFNγ, IL6, TIMP, and MMPs were secreted in a larger amount in culture supernatants of HCEC with EMT-like transition than in those of HCEC w/o the transition; a tendency which was also confirmed by RT-PCR of the corresponding HCECs. Integral PCR array assay of gene signatures relating to EMT-like transition, fibrosis revealed the significant decrease in the expression of distinct genes.
Features of miR signatures of HCEC, either inside cells or secreted form, varied sharply during culture, indicating miR expression fragility. EMT-like transition during culture triggered significant changes of miR traits and SASP-related products. These findings provide a new concept of cell-to-cell interactions propagating cell degeneration, like as in infection.
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