Lian Cui1,2, Yali Lyu1,2, Xiaoliang Jin3, Yueye Wang1, Xiang Li1, Juan Wang1,2, Jieping Zhang1,2, Zhongzhu Deng2, Nan Yang2, Zixuan Zheng1, Yizheng Guo2, Chao Wang2, Rui Mao2, Jingying Xu1,2, Furong Gao1,2, Caixia Jin1,2, Jingfa Zhang1,2, Haibin Tian1,2, Guo-Tong Xu1,2,4,5, Lixia Lu1,2. 1. Department of Ophthalmology, Shanghai Tenth People's Hospital, and Tongji Eye Institute, Tongji University School of Medicine, Shanghai 200072, China. 2. Laboratory of Clinical Visual Science, Department of Regenerative Medicine, and Stem Cell Research Center, Tongji University School of Medicine, Shanghai 200092, China. 3. Department of Ophthalmology, Shanghai Ninth People's Hospital, Shanghai Jiaotong University Medical school, Shanghai 200011, China. 4. Translational Medical Center for Stem Cell Therapy, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 310000, China. 5. The collaborative Innovation Center for Brain Science, Tongji University, Shanghai 310000, China.
Abstract
BACKGROUND: Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of proliferative vitreoretinopathy (PVR). Some microRNAs (miRNAs) participate in regulating RPE cell EMT as post-transcriptional regulators. However, the function of miR-194 in RPE cell EMT remains elusive. Here, the role of miR-194 in PVR was investigated. METHODS: Retinal layers were obtained using laser capture microdissection (LCM). Gene expression at the mRNA and protein level in the tissues and cells was examined using quantitative reverse transcription (RT)-polymerase chain reaction and Western blotting, respectively. The related protein expression was observed by immunostaining. The effect of miR-194 on RPE cell EMT was examined by gel contraction, wound healing, and cell migration assays. RNAseq was performed in ARPE-19 with transfection of pSuper-scramble and pSuper-miR-194. The target gene of miR-194 was identified and confirmed via bioinformatics analysis and dual-luciferase reporter assay. ARPE-19 (adult retinal pigment epithelium-19) cells were treated with transforming growth factor (TGF)-β1 in the same fashion as the in vitro RPE cell EMT model. A PVR rat model was prepared by intravitreous injection of ARPE-19 cells with plasma-rich platelets. RESULTS: miR-194 was preferentially expressed in the RPE cell layer compared with the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer in rat retina. RNAseq analysis indicated that miR-194 overexpression was involved in RPE cell processes, including phagocytosis, ECM-receptor interaction, cell adhesion molecules, and focal adhesion. miR-194 overexpression significantly inhibited the TGF-β1-induced EMT phenotype of RPE cells in vitro. Zinc finger E-box binding homeobox 1 (ZEB1), a key transcription factor in EMT, was confirmed as the direct functional target of miR-194. Knockdown of ZEB1 attenuated TGF-β1-induced α-smooth muscle actin expression in ARPE-19 cells, and overexpression of miR-194 could significantly reduce the expression of some genes which were up-regulated by ZEB1. Exogenous miR-194 administration in vivo effectively suppressed PVR in the rat model, both functionally and structurally. CONCLUSIONS: Our findings demonstrate for the first time that miR-194 suppresses RPE cell EMT by functionally targeting ZEB1. The clinical application of miR-194 in patients with PVR merits further investigation. 2019 Annals of Translational Medicine. All rights reserved.
BACKGROUND: Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of proliferative vitreoretinopathy (PVR). Some microRNAs (miRNAs) participate in regulating RPE cell EMT as post-transcriptional regulators. However, the function of miR-194 in RPE cell EMT remains elusive. Here, the role of miR-194 in PVR was investigated. METHODS: Retinal layers were obtained using laser capture microdissection (LCM). Gene expression at the mRNA and protein level in the tissues and cells was examined using quantitative reverse transcription (RT)-polymerase chain reaction and Western blotting, respectively. The related protein expression was observed by immunostaining. The effect of miR-194 on RPE cell EMT was examined by gel contraction, wound healing, and cell migration assays. RNAseq was performed in ARPE-19 with transfection of pSuper-scramble and pSuper-miR-194. The target gene of miR-194 was identified and confirmed via bioinformatics analysis and dual-luciferase reporter assay. ARPE-19 (adult retinal pigment epithelium-19) cells were treated with transforming growth factor (TGF)-β1 in the same fashion as the in vitro RPE cell EMT model. A PVR rat model was prepared by intravitreous injection of ARPE-19 cells with plasma-rich platelets. RESULTS: miR-194 was preferentially expressed in the RPE cell layer compared with the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer in rat retina. RNAseq analysis indicated that miR-194 overexpression was involved in RPE cell processes, including phagocytosis, ECM-receptor interaction, cell adhesion molecules, and focal adhesion. miR-194 overexpression significantly inhibited the TGF-β1-induced EMT phenotype of RPE cells in vitro. Zinc finger E-box binding homeobox 1 (ZEB1), a key transcription factor in EMT, was confirmed as the direct functional target of miR-194. Knockdown of ZEB1 attenuated TGF-β1-induced α-smooth muscle actin expression in ARPE-19 cells, and overexpression of miR-194 could significantly reduce the expression of some genes which were up-regulated by ZEB1. Exogenous miR-194 administration in vivo effectively suppressed PVR in the rat model, both functionally and structurally. CONCLUSIONS: Our findings demonstrate for the first time that miR-194 suppresses RPE cell EMT by functionally targeting ZEB1. The clinical application of miR-194 in patients with PVR merits further investigation. 2019 Annals of Translational Medicine. All rights reserved.
Authors: Rajdeep Das; Philip A Gregory; Rayzel C Fernandes; Iza Denis; Qingqing Wang; Scott L Townley; Shuang G Zhao; Adrienne R Hanson; Marie A Pickering; Heather K Armstrong; Noor A Lokman; Esmaeil Ebrahimie; Elai Davicioni; Robert B Jenkins; R Jeffrey Karnes; Ashley E Ross; Robert B Den; Eric A Klein; Kim N Chi; Hayley S Ramshaw; Elizabeth D Williams; Amina Zoubeidi; Gregory J Goodall; Felix Y Feng; Lisa M Butler; Wayne D Tilley; Luke A Selth Journal: Cancer Res Date: 2016-12-23 Impact factor: 12.701
Authors: R H Y Asaria; C H Kon; C Bunce; C S Sethi; G A Limb; P T Khaw; G W Aylward; D G Charteris Journal: Br J Ophthalmol Date: 2004-11 Impact factor: 4.638