Regulation of Matrix Metalloproteinase-2 Secretion from Scleral Fibroblasts and Retinal Pigment Epithelial Cells by miR-29a

Abstract

Purpose: To identify an effective method to prevent myopia progression by characterizing the regulation of matrix metalloproteinase- (MMP-) 2 expression and its secretion from scleral fibroblasts and retinal pigment epithelium (RPE) cells by miR-29a.

Methods: The effects of miR-29a on the growth of scleral fibroblasts and RPE cells were assessed using the cell counting kit-8. The changes in MMP-2 mRNA levels in scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor were measured by quantitative PCR. Enzyme-linked immunosorbent assays were used to determine the changes in MMP-2 secretion from scleral fibroblasts and RPE cells after transfection with miR-29a mimics or inhibitor.

Results: The miR-29a mimics or inhibitor did not significantly alter the growth of scleral fibroblasts or RPE cells at 24, 48, or 72 hours after transfection. MMP-2 mRNA levels were significantly decreased in scleral fibroblasts and RPE cells transfected with the miR-29a mimics. The secretion of MMP-2 by scleral fibroblasts and RPE cells was significantly decreased in cells transfected with the miR-29a mimics.

Conclusions: Suppression of scleral fibroblast and RPE cell expression and secretion of MMP-2 by miR-29a can be used as a therapeutic target for the prevention and treatment of myopia.

Figure 1

The efficiency of miR-29a transfection of scleral fibroblasts and retinal pigment epithelial (RPE) cells. Scleral fibroblasts and RPE cells were grown in six-well plates to 60–70% confluency before transfection. The cells in each well were transfected with 50 nM miR-29a mimics or 50 nM miR-29a inhibitor. The quantitative PCR results showed that, in scleral fibroblasts (a) and RPE cells (b), miR-29a was significantly upregulated by transfection of the miR-29a mimics and downregulated by transfection of the miR-29a inhibitor compared with negative controls (P < 0.05). Note: the error bars show the standard deviations (n = 3). The statistical analyses were performed using one-way ANOVA, ^∗P < 0.05. NC, negative controls.

Figure 2

The effects of miR-29a on the viability and growth of scleral fibroblasts and retinal pigment epithelial (RPE) cells. Scleral fibroblasts and RPE cells were seeded at a final density of 3 × 10³/well and cultured in 96-well plates. The cells in each well were transfected with 50 nM miR-29a mimics or 50 nM inhibitor. The miR-29a mimics or inhibitor had no significant effect on the growth of scleral fibroblasts (a) or RPE cells (b) at 24, 48, or 72 hours after transfection compared with the negative controls (P > 0.05). Note: the error bars show the standard deviations (n = 3). The statistical analyses were performed using one-way ANOVA. NC, negative control.

Figure 3

Changes in matrix metalloproteinase- (MMP-) 2 mRNA levels in scleral fibroblasts and retinal pigment epithelial (RPE) cells after transfection with miR-29a mimics or inhibitor. Scleral fibroblasts and RPE cells were grown in six-well plates to 60–70% confluency before transfection. The cells in each well of a six-well plate were transfected with 50 nM mimics or 50 nM inhibitor. Quantitative PCR analyses showed that transfection of the miR-29a mimics decreased MMP2 expression in scleral fibroblasts (a) (P < 0.05) and RPE cells (b) (P < 0.01), while the inhibitor increased MMP-2 expression in scleral fibroblasts (a) (P < 0.05) and RPE cells (b) (P < 0.01) 24 hours after transfection compared with negative controls. Note: the error bars show the standard deviations (n = 3). The statistical analyses were performed using one-way ANOVA. ^∗P < 0.05, ^∗∗P < 0.01. NC, negative control.

Figure 4

Changes in matrix metalloproteinase- (MMP-) 2 protein levels in scleral fibroblasts (a) and retinal pigment epithelial (RPE) cells (b) after transfection with miR-29a mimics or inhibitor. The scleral fibroblasts and RPE cells were grown in six-well plates to 60–70% confluence before transfection. The cells in each well were transfected with 50 nM mimics or 50 nM inhibitor. Western blots showed that the miR-29a mimics decreased MMP2 expression, and the inhibitor increased MMP-2 expression in both scleral fibroblasts and RPE cells 24 hours after transfection. NC, negative control.

Figure 5

Changes in matrix metalloproteinase- (MMP-) 2 secretion from scleral fibroblasts and retinal pigment epithelial (RPE) cells after transfection with miR-29a mimics or inhibitor. The protein secreted from scleral fibroblasts and RPE cells was analyzed using enzyme-linked immunosorbent assays (ELISAs). The secretion of MMP-2 by scleral fibroblasts after 48 hours was decreased in cells transfected with the miR-29a mimics compared with negative controls (P < 0.05). The secretion of MMP-2 by scleral fibroblasts after 72 hours was significantly increased in cells transfected with the miR-29a inhibitor compared with negative controls (P < 0.01). The secretion of MMP-2 was higher 72 hours after transfection than 48 hours after transfection in both the miR-29a mimics group (P < 0.05) and miR-29a inhibitor group (P < 0.01). (a) The secretion of MMP-2 by RPE cells was significantly decreased after 48 (P < 0.01) and 72 (P < 0.05) hours in cells transfected with the miR-29a mimics compared with negative controls. The secretion of MMP-2 by RPE cells after 48 hours was significantly increased in cells transfected with the miR-29a inhibitor compared with negative controls (P < 0.05). The secretion of MMP-2 was higher 72 hours after transfection than 48 hours after transfection in both the miR-29a mimics group (P < 0.01) and miR-29a inhibitor group (P < 0.05) (b). Note: the error bars show the standard deviations (n = 3). The statistical analyses were performed using one-way ANOVA for groups at same time point and paired t-tests for between-group analyses at different time points. ^∗P < 0.05, ^∗∗P < 0.01. NC, negative control; h, hour.