微小RNA-29b过表达降低人角膜内皮细胞中细胞外基质的mRNA和蛋白质生成量。
MicroRNA-29b Overexpression Decreases Extracellular Matrix mRNA and Protein Production in Human Corneal Endothelial Cells.
作者信息
Toyono Tetsuya, Usui Tomohiko, Villarreal Guadalupe, Kallay Laura, Matthaei Mario, Vianna Lucas M M, Zhu Angela Y, Kuroda Masahiko, Amano Shiro, Jun Albert S
机构信息
*Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, MD; †Department of Ophthalmology, University of Tokyo, Tokyo, Japan; ‡Department of Ophthalmology, University of Cologne, Cologne, Germany; §Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan; and ¶Inouye Eye Hospital, Tokyo, Japan.
出版信息
Cornea. 2016 Nov;35(11):1466-1470. doi: 10.1097/ICO.0000000000000954.
PURPOSE
MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. We reported that levels of microRNA (miR)-29 family are decreased in corneas of patients with Fuchs endothelial corneal dystrophy (FECD). The miR-29 family regulates the production of extracellular matrix (ECM) proteins. Accumulation of ECM proteins in Descemet membrane is an important pathologic change in FECD. In this study, we transfected miR-29b into human corneal endothelial cells and tissues and evaluated ECM protein expression levels.
METHODS
An immortalized Fuchs human corneal endothelial cell line (iFECD) was established by infection of corneal endothelial cells from patients with FECD with hTERT lentivirus. MiR-29b was transfected into iFECD, and the expression levels of ECMs collagen type 1 alpha 1 (COL1A1), collagen type 4 alpha 1 (COL4A1), and laminin gamma 1 (LAMC1) were evaluated with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot. Expression level of LAMC1 protein in miR-29b-transfected donor corneal endothelium was also evaluated by Western blot.
RESULTS
Compared with control, miR-29b expression level after transfection of iFECD was increased to 335.6% (±91.0%), and ECM expression levels were significantly decreased. Compared with control, qRT-PCR demonstrated reduction of ECM to the following levels: COL1A1: 1.9% (±0.4%); COL4A1: 7.1% (±1.7%); and LAMC1: 21.5% (±2.7%). Western blot showed reduced protein expression: COL1A1: 4.8% (±3.2%); COL4A1: 42.5% (±25.0%); and LAMC1: 44.8% (±3.1%). In miR-29b-transfected corneal tissue, LAMC1 protein expression level was decreased to 14.4% (±20.5%).
CONCLUSIONS
Overexpression of miR-29b decreased ECM protein production in human corneal endothelial cells. Thus, miR-29 replacement therapy might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet membrane.
目的
微小RNA是一类小的非编码RNA，可在转录后水平调控基因表达。我们报道过，在富克斯内皮性角膜营养不良（FECD）患者的角膜中，微小RNA（miR）-29家族的水平降低。miR-29家族调控细胞外基质（ECM）蛋白的产生。Descemet膜中ECM蛋白的积累是FECD的一项重要病理变化。在本研究中，我们将miR-29b转染到人角膜内皮细胞和组织中，并评估ECM蛋白的表达水平。
方法
通过用hTERT慢病毒感染FECD患者的角膜内皮细胞，建立了永生化的富克斯人角膜内皮细胞系（iFECD）。将miR-29b转染到iFECD中，并用定量逆转录聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法评估ECM的1型胶原α1（COL1A1）、4型胶原α1（COL4A1）和层粘连蛋白γ1（LAMC1）的表达水平。还通过蛋白质免疫印迹法评估了miR-29b转染的供体角膜内皮中LAMC1蛋白的表达水平。
结果
与对照组相比，iFECD转染后miR-29b表达水平提高到335.6%（±91.0%），ECM表达水平显著降低。与对照组相比，qRT-PCR显示ECM降低至以下水平：COL1A1：1.9%（±0.4%）；COL4A1：7.1%（±1.7%）；LAMC1：21.5%（±2.7%）。蛋白质免疫印迹法显示蛋白表达降低：COL1A1：4.8%（±3.2%）；COL4A1：42.5%（±25.0%）；LAMC1：44.8%（±3.1%）。在miR-29b转染的角膜组织中，LAMC1蛋白表达水平降低至14.4%（±20.5%）。
结论
miR-29b的过表达降低了人角膜内皮细胞中ECM蛋白的产生。因此，miR-29替代疗法可能是一种针对FECD的新治疗策略，旨在减少Descemet膜中ECM蛋白的病理性产生。
Zotero 插件