MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells.
MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells.
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DOI：
10.1167/iovs.16-19804
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发表时间：
2016-10
影响因子：
4.4
通讯作者：
Morio Ueno;Kazuko Asada;Munetoyo Toda;Asako Hiraga;Monty M Montoya;C. Sotozono;S. Kinoshita;J. Hamuro
中科院分区：
文献类型：
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作者：
Morio Ueno;Kazuko Asada;Munetoyo Toda;Asako Hiraga;Monty M Montoya;C. Sotozono;S. Kinoshita;J. Hamuro
目的建立一种无创性鉴定培养的人角膜内皮细胞（human corneal endothelial cells，cHCECs）的方法。方法通过细胞表面分化簇（cluster-of-differentiation，CD）标记和细胞形态学的观察，验证cHCECs异质亚群（SPs）组成的差异。通过3D-Gene Human microRNA Chips（Toray Industries，Inc.）检测培养细胞或上清液中的microRNA（miRNA）谱。还分析了具有不同内皮细胞密度（ECD）的新鲜角膜组织（含或不含gutatta）的曲线。为了验证3D-Gene结果，进行了定量实时聚合酶链反应（PCR）。从用选择的miRNA转染的cHCEC提取RNA，并通过PCR阵列（Qiagen）推测靶基因。结果在不同形态的cHCECs中，miRNA表达谱存在差异。一种能够区分CD 44- SP与具有CD 44 ++ CD 44+表型的SP的miRNA被鉴定为miR 34 a。378家族中miRNAs的下调导致cHCECs表面CD 44的上调。有趣的是，角膜内皮中378家族中上调的miRNA在具有较低ECD的组织中显著降低，具有晚期gutatta，这为Fuchs角膜内皮营养不良的发病机制提供了新的见解。结论与成熟HCECs共享CD 44表面表型的特定培养SP显示miR-378的最高表达。相反，具有上调的CD 44+的SP显示miR-378减少。因此，培养细胞中的miRNA可以作为鉴定cHCEC的替代方法。
Purpose To elucidate a noninvasive method to qualify and identify cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition and adaptable for cell-based therapy. Methods The variations of cHCECs in their composition of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers and their morphology. The profiles of microRNA (miRNA) in cultured cells or supernatants were detected by 3D-Gene Human microRNA Chips (Toray Industries, Inc.). The profiles were also analyzed for fresh corneal tissues with distinct endothelial cell densities (ECD) with or without gutatta. To validate the 3D-Gene results, quantitative real-time polymerase chain reaction (PCR) was performed. RNAs were extracted from cHCECs transfected with selected miRNA, and target genes were presumed by PCR array (Qiagen). Results Among a variety of morphologically different cHCECs, miRNA expression profiles were distinctively revealed. The one miRNA capable of discriminating CD44- SP from SPs with CD44++∼CD44+++ phenotypes was identified as miR34a. The downregulation of miRNAs in the 378 family paralleled the upregulation of surface CD44 on cHCECs. Interestingly, upregulated miRNAs in the 378 family in corneal endothelium dramatically decreased in the tissues with lower ECD with advanced gutatta, providing new insight on the pathogenesis of Fuchs' endothelial corneal dystrophy. Conclusions The specified cultured SPs sharing the CD44- surface phenotypes with matured HCECs showed the highest expression of miR-378. Conversely, SPs with upregulated CD44+++ showed a reduction of miR-378. Thus, miRNA in cultured cells may serve as an alternative method to qualify cHCECs.