Purchase this article with an account.
Shigeru Kinoshita, Morio Ueno, Kazuko Asada, Munetoyo Toda, Kazue Nagata, Chie Sotozono, Nobuyoshi Kosaka, Takahiro Ochiya, Junji Hamuro; Regulation of mitochondrial respiration under cell culture stress in human corneal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Cultured human corneal endothelial cells (cHCECs) serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction. This study investigated the underlying mechanism of cell-state transition (CST) by utilizing the integral profiles of secreted microRNA (miRNA), exosomes, and metabolites of cHCECs.
An integrated analysis of miRNA profiles in HCEC culture supernatants (CS) was investigated by 3D-Gene® DNA Chip (Toray) microarray. To validate the 3D-Gene® DNA Chip microarray results, quantitative reverse transcriptase polymerase chain reaction was carried out for a vast number of cells cultures, distinct in their morphology and the composites of subpopulations (SPs). The exosomes and miRNAs in the CS were also analyzed. Metabolic extracts in CS of cHCECs were prepared and analyzed using a capillary electrophoresis (CE)-connected CE-MS/MS system (CARCINOSCOPE; HMT Inc.).
The candidate miRNAs to discriminate CD44- SPs from those with CD44++~ CD44+++ phenotypes were miRs 221-3p, 1246, 1915-3p, and 4732-5p. The levels of the latter three miRs decreased dramatically in the CS of cHCECs without CST, compared with those of control medium, while those from cHCECs with senescence-like CST showed an increase. Of note, miR184 only decreased inversely in parallel with the upregulation of CD44 on cHCECs. In contrast with miRs in CS, the only intracellular miR capable to discriminate a CD44- SP from SPs with CD44++~CD44+++ phenotypes was identified as miR-34a. CD9+ exosomes were highly elevated in the CS of cHCECs with senescence-like CST than those without CST, indicating the possible import of these extracellular vesicles into cHCECs without CST. The reduction of glycolysis in matured differentiated cHCECs SPs were evident by integral analysis of the secreted metabolites. This might be triggered by c-Myc pathway down-regulation, the hallmark to distinguish differentiated cHCECs from either non-differentiated or cHCECs with CST.
The cHCECs sharing a CD44- phenotype of matured HCECs may be discriminated by measuring the amount of miRNAs, exosomes, or metabolites in CS. The miR-34a axis may regulate the expression and activation of CD44 and its downstream factors Ras homolog gene family member A and increase a metabolic flux to mitochondrial respiration and concomitantly inhibited entry into glycolysis.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
This PDF is available to Subscribers Only