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Shuai Yang, Haipei Yao, Min Li, Hui Li, Fang Wang; Long non-coding RNA MALAT1 mediates transforming growth factor beta1-induced epithelial to mesenchymal transition in retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5369.
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© ARVO (1962-2015); The Authors (2016-present)
Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a hallmark in the development of proliferative vitreoretinopathy (PVR). Currently, long non-coding RNAs (lncRNAs) are emerging as key regulators in various biological processes. However, the role of lncRNAs in EMT of RPE cells remains largely unknown. This study investigated the role of lncRNA-MALAT1 in human RPE cells upon TGFβ1 induction of EMT.
ARPE-19 cells was cultured and exposed to TGF-β1. EMT was confirmed by morphological change, as well as the increased expression of alpha-smooth muscle actin (αSMA) and fibronectin, and the down-regulation of E-cadherin and Zona occludin-1(ZO-1) at both mRNA and protein levels. Expression of lncRNA MALAT1 was evaluated by real-time PCR (RT-PCR) at 0, 12, 24, 48, and 72 hours after TGFβ1 stimulation. SiRNA targeting MALAT1 was designed and the silencing efficiency was determined by RT-PCR. The biological effect of MALAT1 siRNA on EMT, migration, and viability of RPE cells were evaluated by detecting EMT-related genes, transwell assay and MTS assay, respectively. The effect of MALAT1 silencing on phosphorylation of Smad2/3 and p-38 were detected by western blot. We also determined MALAT1 expression in primary RPE cells incubated with PVR vitreous samples.
MALAT1 is dramatically up-regulated in ARPE-19 cells incubated with TGFβ1 and reaches the apex at 48 hours (fold change=2.80±0.16, P=0.003). Transfection of MALAT1-SiRNA decreased the expression of MALAT1 by 67%. MALAT1 silencing attenuated TGFβ1-induced morphological transition of RPE cells. Western blot, immunofluorescence, and RT-PCR results show that MALAT1 is required for the TGFβ1-induced upregulating of αSMA and fibronectin, as well as down-regulating of ZO-1 and E-Cadherin. Inhibition of MALAT1 attenuates TGFβ1-induced migration and proliferation of RPE cells. Besides, MALAT1 activates the canonical Smad2/3, but not the non-canonical p38 signaling of TGFβ. Furthermore, MALAT1 is increased by 3.14±0.78 times (n=4, P=0.038) in human primary RPE cells incubated with PVR vitreous samples compared with cells exposed to normal vitreous.
LncRNA MALAT1 is involved in TGFβ1-induced EMT of human RPE cells and provided new understanding for the pathogenesis of PVR.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Knockdown of MALAT1 attenuates TGF-β1 induced EMT in RPE cells
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