MicroRNA signature in wound healing following excimer laser ablation: role of miR-133b on TGFβ1, CTGF, SMA, and COL1A1 expression levels in rabbit corneal fibroblasts

Abstract

Purpose: The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes.

Methods: Laser-ablated mouse corneas were collected at 0 and 30 minutes and 2 days. Ribonucleic acid was collected from corneas and analyzed using cell differentiation and development miRNA PCR arrays. Luciferase assay was used to determine whether miR-133b targeted the 3' untranslated region (UTR) of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) in rabbit corneal fibroblasts (RbCF). Quantitative real-time PCR (qRT-PCR) and Western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA), and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration.

Results: At day 2, 37 of 86 miRNAs had substantial expression fold changes. miR-133b had the greatest fold decrease at -14.33. Pre-miR-133b targeted the 3' UTR of CTGF and caused a significant decrease of 38% (P < 0.01). Transforming growth factor β1-treated RbCF had a significant decrease of miR-133b of 49% (P < 0.01), whereas CTGF, SMA, and COL1A1 had significant increases of 20%, 54%, and 37% (P < 0.01), respectively. The RbCF treated with TGFβ1 and pre-miR133b showed significant decreases in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (P < 0.01), respectively. Finally, there was significant decrease in migration of miR-133b-treated RbCF.

Conclusions: Significant changes occur in key miRNAs during early corneal wound healing, suggesting novel miRNA targets to reduce scar formation.

Keywords: CTGF; corneal wound healing; gene expression; microRNA.

Figure 1.

Determination of the ability of miR-133b to target the 3′ UTR of CTGF and TGFβ1. After dual transfection of a luciferase reporter plasmid that contained the 3′ UTR of either CTGF (a) or TGFβ1 (b) and either the pre-NC or the pre-miR-133b into RbCF, relative luciferase activity and significance were determined.

Figure 2.

Endogenous expression levels of miR-133b, CTGF, SMA, and COL1A1 in RbCF. Using qRT-PCR, relative expression levels of miR-133b (a), CTGF (b), SMA (b), and COL1A1 were determined in unstimulated control (con) in RbCF or RbCF stimulated with TGFβ1 (2.5 ng/mL) for 12 (a) and 24 hours (b).

Figure 3.

Effect of anti-miR-133b and miR-133b on mRNA and protein levels of CTGF. (a) Using Western blot, relative expression levels of CTGF protein were determined after 96-hour transfection of prenegative control (NC), pre-miR-133b, anti-NC, and anti-miR-133b. (b) Protein expression levels were quantified by comparing to the α-tubulin control (n = 5). (c) Expression levels of CTGF mRNA from RbCF were quantified using qRT-PCR after 72-hour transfection of NC or pre-miR-133b and TGFβ1 (2.5 ng/mL) treatment for the last 24 hours. (d) Using Western blot, expression levels of CTGF protein were determined after 96-hour transfection of NC or pre-miR-133b and TGFβ1 (2.5 ng/mL) treatment for the last 48 hours. (e) Protein expression levels were quantified by comparing to the α-tubulin control (n = 8).

Figure 4.

Effect of pre-miR-133b on relative expression levels of SMA and COL1A1 in RbCF. Using qRT-PCR, relative expression levels of SMA (a) and COLA1A (b) were determined after 72-hour transfection of NC or pre-miR-133b and TGFβ1 (2.5 ng/mL) treatment for the last 24 hours.

Figure 5.

Effect of pre-miR-133b on migration of RbCF. A migration assay was performed using control RbCF or RbCF stimulated with TGFβ1 or CTGF and treated with either a NC or pre-miR-133b. (a) Cell migration assay of the different treatments. (b) Quantification of the cell migration due to the different treatments (*P < 0.05).