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.
OBJECTIVE
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.
Department of Pediatrics, University of Florida, Gainesville, Florida.
Paulette M
PM
Robinson
Tsai-Der
TD
Chuang
Sriniwas
S
Sriram
Liya
L
Pi
Xiao Ping
XP
Luo
Bryon E
BE
Petersen
Gregory S
GS
Schultz
D000199:Actins / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D024042:Collagen Type I / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000378:metabolism; D004195:Disease Models, Animal; D019573:Epithelium, Corneal / Q000293:injuries* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D054018:Lasers, Excimer / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000378:metabolism*; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology; D014945:Wound Healing / Q000502:physiology*
D000199:Actins; D024042:Collagen Type I; D035683:MicroRNAs; C517732:Mirn133 microRNA, mouse; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor
CTGF; corneal wound healing; gene expression; microRNA