The microRNAs miR-302d and miR-93 inhibit TGFB-mediated EMT and VEGFA secretion from ARPE-19 cells

https://doi.org/10.1016/j.exer.2020.108258Get rights and content

Highlights

  • miR-302d and miR-93 are capable of inhibiting TGFB-mediated VEGFA secretion from ARPE-19 cells by directly targeting TGFBR2, as well as VEGFA.

  • Both miRNAs can prevent TGFB–induced EMT of ARPE-19 cells in vitro, mainly by inhibiting TGFBR2 at the transcriptional and translational level.

  • Both miRNAs can revert TGFB-induced mesenchymal ARPE-19 cells towards an epithelial-like state and therefore promote MET.

Abstract

The transforming growth factor-beta (TGFB) plays an essential role in the pathogenesis of some ophthalmologic diseases, including neovascular age-related macular degeneration (nAMD) and proliferative vitreoretinopathy (PVR). TGFB activates the transcription factors SMAD2 and SMAD3 via the TGFB receptor, which together activate several genes, including VEGFA. TGFB treated ARPE-19 cells show an increased proliferation rate and undergo epithelial to mesenchymal transition (EMT). Since microRNAs (miRNAs) are capable of inhibiting the translation of multiple genes, we screened for miRNAs that regulate the TGFB signalling pathways at multiple levels. In this study, we focused on two miRNAs, miR-302d and miR-93, which inhibit TGFB signalling pathway and therefore TGFB-induced EMT transition as well as VEGFA secretion from ARPE-19 cells. Furthermore, we could show that both miRNAs can retransform TGFB-stimulated mesenchymal ARPE-19 cells towards the morphological epithelial-like state. Taken together, transient overexpression of these miRNAs in RPE cells might be a promising approach for further translational strategies.

Introduction

The Transforming Growth Factor Beta (TGFB) is a multifunctional cytokine that plays an essential role in the pathogenesis of ophthalmologic diseases, such as proliferative vitreoretinopathy (PVR) and the wet form of age-related macular degeneration (AMD). The influence of high levels of TGFB on the retinal pigment epithelium (RPE) is very versatile. Binding of TGFB to TGFB receptor which consists of the subunits TGFBR1 and TGFBR2 induces phosphorylation of SMAD2 and SMAD3. P-Smad2/3 activate their effector genes including the vascular endothelial growth factor A (VEGFA); (Bian et al., 2007; Eichler et al., 2002; Gamulescu et al., 2006; Zeng et al., 2006). VEGFA is a signalling molecule which is released by the RPE cells. It is essential for the maintenance of the choroid vessels, but too high levels of VEGFA can induce the formation of new potentially leaking blood vessels, as in the case for wet AMD. For about 15 years, anti-VEGF therapy for neovascular AMD has become one of the most widely used standard treatments.

An additional well-known effect of TGFB on RPE cells is that it promotes epithelial to mesenchymal transition (EMT); (Gamulescu et al., 2006). EMT causes morphological changes of epithelial cells towards a mesenchymal phenotype which is characterised by (i) reassembly of the radially extending F-actin cytoskeleton to a parallelly orientated array, (ii) directional alignment of the cells which is recognisable as scarring in vivo or vortex-like arrangement in vitro, (iii) lack of tight junction proteins such as ZO-1 and Occludin which are essential to maintain the blood-retina barrier and (iv) increased proliferation and expression of smooth muscle actin which contributes to the contractile property of fibrotic RPE cells subsequently leading to the detachment of the retina in vivo as observed in the pathology of PVR. The contractile detachment of the retina causes cell death of the photoreceptors leading to severe vision loss. Even though surgical techniques have improved over time, and novel antiangiogenic drugs have been established, current therapeutic approaches are improvable to prevent PVR.

Since the TGFB-mediated EMT is characteristic for the pathology of many diseases, including metastatic tumours, different approaches to inhibit the TGFB signalling pathway has been investigated. These approaches included, (i) blocking TGFB receptors with small molecules, (ii) intercepting TGFB itself with modified antibodies and (iii) knockdown of TGFB-signalling-related genes with short interfering RNAs (siRNAs); (Carrington et al., 2000). The blocking of TGFBR1 with the small molecule SB431542, which have been used for this study, was already reported in 2002 (Laping et al., 2002). Galunisertib, another TGFBR1 blocker, has reached phase 2 clinical trials in the treatment of hepatocellular carcinoma (Fransvea et al., 2011; Herbertz et al., 2015). Further therapeutic approaches for short-term treatment without genetic modification are conceivable. An example of such an approach could be achieved by modulating the expression of small RNAs, including short-interference RNAs (siRNAs) or microRNAs (miRNAs); (Chakraborty et al., 2017).

MicroRNAs (miRNAs) were first discovered by Victor Ambros group in 1993 in the nematode C. elegans and belong to a class of small, endogenous, evolutionarily conserved RNAs of about 19–24 nucleotide length (Lee et al., 1993). MiRNAs inhibit protein synthesis by binding to semi-complementary sequences in the 3′-UTR of their target messenger RNAs. The fact that miRNAs inhibit their target genes due to imperfect binding, miRNA target-gene prediction tools such as PicTAR, miRDB and Targetscan were developed based on experimental results and specific algorithms (Agarwal et al., 2015; Krek et al., 2005; Lewis et al., 2005; Wong and Wang, 2015). To date, more than 2000 different miRNAs have been reported in humans that are presumed to regulate about 1/3rd of all mRNAs post-transcriptionally directly. Further studies show that miRNAs not only restrict the translation of their target mRNAs but also promote their destabilisation leading to reduced mRNA expression levels (Eichhorn et al., 2014).

MicroRNA is also the focus of many ophthalmological studies. MicroRNA expression analyses were performed on vitreous humour fluids from patients with diabetic retinopathy or glaucoma (Chen et al., 2019; Jayaram et al., 2017; Martinez and Peplow, 2019). These data might be useful to identify microRNAs as biomarkers for the early detection of eye diseases as well as initiate appropriate therapeutic approaches as early as possible. Other studies concentrated on the influence of microRNAs on the pathogenesis of PVR or AMD, with a focus on EMT of RPE cells. For example, it has been reported that miR-124 inhibits TGFB-mediated EMT of the RPE cell line ARPE-19 in vitro by targeting RHOG (Jun and Joo, 2016). Another group demonstrated that miR-30b expression is upregulated by hydrogen peroxide-induced oxidative stress, and it potentially downregulates the antioxidant enzyme catalase in ARPE-19 cells. Catalase activity is essential to prevent RPE cells from oxidative stress, a key factor for the pathogenesis of AMD. The authors hypothesised that blocking miR-30b with antisense oligos could prevent miR-30b-mediated degradation of catalase and therefore, could protect RPE cells from oxidative stress (Haque et al., 2012).

The aim of our in vitro study presented here was to identify miRNAs that were capable to (i) inhibit TGFB-mediated EMT, and (ii) reduce TGFB-mediated secretion of VEGFA from the retinal pigment epithelial cell line ARPE-19. Both events, the TGFB-Mediated EMT and VEGFA secretion play a role in the pathogenesis of PVR and wet AMD.

Here, we report that overexpression of miR-93 or miR-302d can prevent TGFB-mediated EMT as well as VEGFA secretion of ARPE-19 cells due to inhibition of the TGFBR2 and VEGFA itself. Moreover, we show that both miRNAs were even capable of reverting TGFB-induced mesenchymal ARPE-19 cells towards an epithelial-like state. Since excessive VEGF secretion, as well as fibrosis of the RPE, is part of various ophthalmological diseases, the overexpression of miR-302d or miR-93 would be a possible approach for translational therapies.

Section snippets

Cell culture

The human RPE cell line ARPE-19 was grown in DMEM/F12 media (Gibco #21331-020) supplemented with 1X GlutaMAX™(Gibco #3505-061), 10% FBS (Pan-Biotech #P30-3606) and 2% Pen-Strep (Gibco #15140-122). For TGFB treatments and miRNA transfection, the same media was used but without Pen-Strep and 1% FBS.

microRNA gene target validation

One day before transfection, 5 × 104 ARPE-19 cells were seeded into each well of a 12-well plate (Sarstedt #83.3922.300) in 1 mL ARPE-19 expansion media. Thirty minutes before transfection, media was

Results

MicroRNAs exhibit pleiotropic effects on gene expression. Therefore, we wanted to investigate whether the miRNAs potentially regulating multiple genes in a signalling cascade could be used for translational purposes. For this approach, we chose the TGFB signalling pathway, which plays a role in the pathogenesis of many diseases, including proliferative retinopathy of RPE cells. Activation of TGFB-signalling by TGFB1 or TGFB2 has been reported to (i) induce Epithelial to Mesenchymal Transition

Discussion

To date, several cytokines have been identified in vitreous fluids that are elevated in PVR patients and could trigger EMT. These include fibroblast growth factor (FGF), vascular endothelial factor (VEGF) as well as transforming growth factor-beta (TGFB); (Banerjee et al., 2007; Hoerster et al., 2013, 2014). TGFB, which consists of three different isoforms in humans (TGFB1,2,3), seems to be one of the most potent initiators of EMT. In the aqueous humour, TGFB2 is one of the most predominantly

Author contributions

HF, CF designed the experiments. HF, RM conducted experiments. HF, RL, CF wrote the manuscript.

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      Downstream of these, cytokines, chemokines, and growth factors (including FGF and TGF-β) are released (reviewed in Chaudhary et al., 2020). Multiple in vitro studies highlight the role of TGF-β signaling on EMT and promoting proliferation of the fibroblast-like RPE cells, and several recent studies have identified a novel role for miRNAs, including miR-124, miR-302d, and miR-93, in inhibiting TGF-β-mediated EMT and the maintenance of fibroblastic phenotypes in cultured RPE cells (Jun and Joo, 2016; Fuchs et al., 2020). Importantly, transfection of miR-302d and miR-93 retransformed fibroblast-like RPE cells back to an epithelial-like state, regardless of TGF-β exposure, as well as inhibited the TGF-β-mediated secretion of VEGF (Fuchs et al., 2020).

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