Knockdown of survivin results in inhibition of epithelial to mesenchymal transition in retinal pigment epithelial cells by attenuating the TGFβ pathway

Abstract

Proliferative vitreoretinopathy (PVR) is a common complication of open globe injury and the most common cause of failed retinal detachment surgery. The response by retinal pigment epithelial (RPE) cells liberated into the vitreous includes proliferation and migration; most importantly, epithelial to mesenchymal transition (EMT) of RPE plays a central role in the development and progress of PVR. For the first time, we show that knockdown of BIRC5, a member of the inhibitor of apoptosis protein family, using either lentiviral vector based CRISPR/Cas9 nickase gene editing or inhibition of survivin using the small-molecule inhibitor YM155, results in the suppression of EMT in RPE cells. Knockdown of survivin or inhibition of survivin significantly reduced TGFβ-induced cell proliferation and migration. We further demonstrated that knockdown or inhibition of survivin attenuated the TGFβ signaling by showing reduced phospho-SMAD2 in BIRC5 knockdown or YM155-treated cells compared to controls. Inhibition of the TGFβ pathway using TGFβ receptor inhibitor also suppressed survivin expression in RPE cells. Our studies demonstrate that survivin contributes to EMT by cross-talking with the TGFβ pathway in RPE cells. Targeting survivin using small-molecule inhibitors may provide a novel approach to treat PVR disease.

Keywords: BIRC5; Epithelial to mesenchymal transition (EMT); Lentiviral CRISPR/Cas9 nickase vector; Retinal pigment epithelial cells; Survivin; YM155.

Figure 1. Knockdown or inhibition of survivin suppresses EMT in ARPE-19 cells

A. Western blot analysis of survivin and EMT markers in survivin knockdown(KD) and control ARPE-19 cells generated using lentiviral CRISPR/Cas9nickase vector. B. and C. Immunofluorescent staining of mesenchymal marker β-catenin (B) and epithelial marker cytokeratin-7 (C) in survivin KD and control ARPE-19 cells. D. Western blot analysis of EMT markers in wildtype ARPE-19 cells at 48 h following different doses of survivin inhibitor YM155 treatment. E. Western blot analysis of EMT markers at different time points following 20 nM YM155 treatment.

Figure 2. Knockdown or inhibition of survivin suppresses TGFβ-induced cell proliferation

A. Cell proliferation in survivin KD and control cells was measured by MTT assay at different time points following 6 ng/ml TGFβ treatment (*p<0.05). B. Cell proliferation was measured using MTT assay following 20 nM YM155 treatment for 4 h and then treated with 6 ng/ml TGFβ(*p<0.05,**p<0.01,***p<0.001).

Figure 3. Knockdown or inhibition of survivin suppresses TGFβ-induced cell migration

A. Cell migration in survivin KD and control cells was examined using transwell plates at different time points following 6 ng/ml TGFβ treatment(*p<0.05;**p<0.01). B. Cell migration was measured using transwell plates following 20 nM YM155 treatment for 4 h and then treated with 6 ng/ml TGFβ (*p<0.05;**p<0.01).

Figure 4. Survivin contributes to EMT by activating TGFβ pathways

A. Western blot analysis of survivin expression following different doses of TGFβ treatment for 24 h (**p<0.01;***p<0.001). B. Western blot analysis of survivin (*p<0.05) and phospho-SMAD2 (***p<0.001) expression following 20 µM TGFβ inhibitor and 6 ng/ml TGFβ treatment. C. Western blot analysis of phospho-SMAD2 (*p<0.05;**p<0.01) and total SMAD2/3 at different time points in survivin KD and control cells following 6 ng/ml TGFβ treatment. D. Western blot analysis of phospho-SMAD2 (**p<0.01) and total SMAD2/3 in wildtype ARPE-19 cells following 20 nM YM155 treatment for 4 h, then treated with 6 ng/ml TGFβ. E. Survivin KD and control cell morphology following 6 ng/ml TGFβ treatment for 24 h. F. Wildtype ARPE-19 cell morphology at 24 h following 20 nM YM155 treatment and then with 6 ng/ml TGFβ treatment.