miR-194 suppresses epithelial-mesenchymal transition of retinal pigment epithelial cells by directly targeting ZEB1

Lian Cui, Yali Lyu, Xiaoliang Jin, Yueye Wang, Xiang Li, Juan Wang, Jieping Zhang, Zhongzhu Deng, Nan Yang, Zixuan Zheng, Yizheng Guo, Chao Wang, Rui Mao, Jingying Xu, Furong Gao, Caixia Jin, Jingfa Zhang, Haibin Tian, Guo-Tong Xu, Lixia Lu


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.