Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells

Abstract

Purpose: To study the role of long non-coding RNA (lncRNA) MALAT1 in transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells.

Methods: ARPE-19 cells were cultured and exposed to TGF-β1. The EMT of APRE-19 cells is 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. The expression of lncRNA MALAT1 in RPE cells were detected by quantitative real-time PCR. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA). The effect of inhibition of MALAT1 on EMT, migration, proliferation, and TGFβ signalings were observed. MALAT1 expression was also detected in primary RPE cells incubated with proliferative vitreoretinopathy (PVR) vitreous samples.

Results: The expression of MALAT1 is significantly increased in RPE cells incubated with TGFβ1. MALAT1 silencing attenuates TGFβ1-induced EMT, migration, and proliferation of RPE cells, at least partially through activating Smad2/3 signaling. MALAT1 is also significantly increased in primary RPE cells incubated with PVR vitreous samples.

Conclusion: LncRNA MALAT1 is involved in TGFβ1-induced EMT of human RPE cells and provides new understandings for the pathogenesis of PVR.

Fig 1. TGF-β1 induces EMT and MALAT1 expression in ARPE-19 cells.

APRE-19 cells were incubated with TGF-β1 (10 ng/ml) for 48h. A. TGF-β1 induces the morphological change of APRE-19 cells were captured at 100× magnification. B-D. The expression level of EMT-related markers (E-Cadherin, ZO-1, α-SMA, and Fibronection) were detected by immunofluorescence, RT-PCR, and Western blot. E. Quantification of the relative protein expression (normalized to β-actin) in western blot. F. The expression of MALAT1 was detected by RT-PCR in indicated time points.

Fig 2. Knockdown of MALAT1 attenuates the TGF-β1 induced EMT in RPE cells.

ARPE-19 cells were transfected with MALAT1 SiRNA (Si-MALAT1) or negative control SiRNA (Si-NC) and were treated with or without TGF-β1 (10ng/ml) for 48 h. A. The expression levels of MALAT1 were detected by RT-PCR. B-C. The expression level of EMT-related markers (E-Cadherin, ZO-1, α-SMA, and Fibronection) were detected by RT-PCR and Western blot. D, Quantification of the relative protein expression (normalized to β-actin) in C. E. The morphologic appearances of the cells were captured at 100× magnification. F. The expression of E-Cadherin, ZO-1, α-SMA, and Fibronection werer detected by immunofluorescence.

Fig 3. Knocking-down of MALAT1 reduced the TGF-β1-induced up-regulation of Snail, SLUG, and ZEB1 in RPE cells.

A-D ARPE-19 cells were transfected with MALAT1 SiRNA (Si-MALAT1) or negative control SiRNA (Si-NC) and were treated with or without TGF-β1 (10ng/ml) for 48 h. The expression level of EMT-related transcription factors (Snail, SLUG, and ZEB1) were detected and quantified by RT-PCR (A), Western blot (B-C) and immunofluorescence (D).

Fig 4. Downregulation of MALAT1 inhibited TGF-β1 induced migration and proliferation in RPE cells.

A. ARPE-19 cells were transfected with MALAT1 SiRNA (Si-MALAT1) or negative control SiRNA (Si-NC) and were treated with or without TGF-β1 (10ng/ml) for 48 h. Cells were then subjected to transwell migration assay. B. The number of migrated cells was quantified by counting 5 random vision fields in a microscope (magnification: ×200). C. ARPE-19 cells were transfected with Si-MALAT1 or Si-NC. A scratch was then made to the cell monolayer and TGF-β1 (10ng/ml) was applied. Photographs were taken at indicated times. D. The ratios of remaining of gap at 48 h were calculated. E. After siRNA transfection and TGF-β1 incubation, cell proliferation was assessed using an MTT cell proliferation assay kit.

Fig 5. Downregulating MALAT1 inhibits the phosphorylation of Smad2/3.

ARPE-19 cells transfected with Si-MALAT1 or Si-NC were treated with TGF-β1 (10 ng/ml) for 1 h. The expression of p-Smad2/3 and Smad2/3 (A), as well as p-38 and p38 (C) were examined by western blot. The relative protein expressions were quantified by normalizing to the GAPDH expression (B and D).

Fig 6

MALAT1 expression is increased in human primary RPE cells incubated TGF-β1 (10 ng/ml) (A) or PVR vitreous (B) as detected by RT-PCR.