Abstract
Purpose :
The transforming growth factor beta (TGFB) plays an important role in the pathogenesis of a number of ophthalmologic diseases, including glaucoma and proliferative vitreoretinopathy. TGFB activates the transcription factors SMAD2 and SMAD3 via the TGFB receptor, which together activates a number of genes, including VEGFA and MMP2. 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 signaling pathways at multiple levels. In this study, we focused on two miRNAs inhibiting TGFB signaling pathway as well as TGFB-induced EMT transition of ARPE-19 cells.
Methods :
microRNA target genes were evaluated by using a luciferase-based reporter construct. ARPE-19 cells have been treated with 10-20ng/ml recombinant human TGFB and transfected with neg. control miRNA, miR-199 or miR-17. Expression of TGFB-signaling associated genes, such as TGFB, TGFBR2, SMAD2, SMAD3 and downstream targets such as MMP2 and VEGFA was analyzed by RT-PCR. ICC-staining for ZO-1 and VIM was performed after 7 days of TGFB1 treatment. P-SMAD2 protein levels were quantified by WB after 60min of TGFB-Treatment. VEGF secretion has been measured after TGFB stimulation by ELISA.
Results :
72h after miRNA transfection and TGFB treatment, the control transfected cells show morphological changes towards a mesenchymal phenotype, whereas the miR-199 or miR-17 transfected cells remained an epithelial-like state. Using the luciferase-based miRNA-target gene construct, we confirm TGFB1, TGFBR2, SMAD2, MMP2, and VEGFA as target genes for miR-199 and miR-17. The RT-PCR analysis shows that miR-199 and miR-17 inhibit the expression of TGFB associated genes (e.g. MMP2, VEGF, VIM) and promote the expression of epithelial markers such as CDH1. Western blot analysis reveals that P-SMAD2 protein levels are strongly reduced by miR-17 and miR-199 after 1h of TGFB treatment. Furthermore, TGFB-induced VEGFA secretion is significantly reduced by miR-199 and miR-17 compared to the control transfection.
Conclusions :
Our data show that miR-199 and miR-17 are capable of preventing a TGFB-induced EMT transition. Furthermore, our data indicate that both miRNAs inhibit the secretion of VEGF and MMP2, two genes that are in focus of various retinal diseases.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.