Effect of SOX2 Repression on Corneal Endothelial Cells

Abstract

Purpose: Human corneal endothelial cells (hCECs) pump out water from the stroma and maintain the clarity of the cornea. The sex-determining region Y-box 2 (SOX2) participates in differentiation during the development of the anterior segment of the eye and is found in the periphery of wounded corneas. This study was performed to investigate the effect of SOX2 repression on hCECs.

Methods: Cultured hCECs were transfected by siRNA for SOX2. The wound healing rate and cell viability were measured. The cell proliferation-associated protein level was evaluated by Western blotting and RT-PCR. The energy production and mitochondrial function were measured, and cell shape and WNT signaling were assessed.

Results: Upon transfecting the cultured cells with siRNA for SOX2, the SOX2 level was reduced by 80%. The wound healing rate and viability were also reduced. Additionally, CDK1, cyclin D1, SIRT1, and ATP5B levels were reduced, and CDKN2A and pAMPK levels were increased. Mitochondrial oxidative stress and mitochondrial viability decreased, and the cell shape became elongated. Furthermore, SMAD1, SNAI1, WNT3A, and β-catenin levels were increased.

Conclusion: SOX2 repression disrupts the normal metabolism of hCECs through modulating WNT signaling and mitochondrial functions.

Keywords: SOX2; WNT signaling; human corneal endothelial cells.

Figure 1

Human corneal endothelial cell (hCEC) culture and transfection of siRNA. (A) hCECs cultured at P0. The scale bar denotes 200 μm. (B) Green fluorescence of transfected cells indicates fluorescein isothiocyanate-conjugated control siRNA at 48 h after transfection. The confluency of the cells at the time of transfection should be about 70%. The scale bar denotes 100 μm. (C) Immunostaining for zonula occludens-1 (ZO-1). Green is ZO-1 and blue is Hoechst 33,342 nuclear staining. The scale bar denotes 50 μm. (D) Immunostaining for zonula connexin 43 (Cx43). Green is Cx43 and blue is Hoechst 33,342 nuclear staining. The scale bar denotes 50 μm. (E,F) sex-determining region Y-box 2 (SOX2) level was evaluated using RT-PCR and Western blotting. (G) Scratch assay monitoring the wound healing rate. Wound healing was delayed in si-SOX2-transfected cells. * indicates statistical significance by independent t-test.

Figure 2

Endothelial-mesenchymal transition and WNT signaling pathway. (A) Cell shape in si-control and si-SOX2 cells. Scale bar denotes 150 μm. (B) SMAD1 mRNA expression. (C) α-SMA level determined by Western blotting. (D) SNAI1 level determined by Western blotting. (E) WNT3A mRNA expression. (F) GSK3β activation determined by Western blotting. (G,H) β-catenin level determined by RT-PCR and Western blotting. * indicates statistical significance by independent t-test.

Figure 3

Cell viability and proliferation. (A) Cell viability. (B) BrdU cell proliferation rate. (C) Cell cycle analysis. (D) CDK1 and cyclin D1 levels. (E) CDKN2A mRNA expression evaluated by RT-PCR. (F) CDKN2A level evaluated by Western blotting was higher in si-SOX2-transfected cells. Graph provides the results from triple experiments. * indicates statistical significance by independent t-test.

Figure 4

Mitochondrial functions. (A) Energy production in hCECs. (B) Mitochondrial membrane potential using the JC-1 probe. Scale bar denotes 100 μm. (C) Mitochondrial viability. Scale bar denotes 75 μm. Arrows indicating nuclei. (D) pAMPK level. (E) SIRT1 level. (F) ATP5B amount was lower in si-SOX2-transfected cells. Graph provides the results from triple experiments. (G) Mitochondrial oxidative stress levels by MitoSOX fluorescence intensity. Scale bar denotes 50 μm. * indicates statistical significance by independent t-test.