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Paulette Robinson, Tsai-Der Chuang, Sriniwas Sriram, Liya Pi, Xiao Ping Luo, Bryon Petersen, Gregory Schultz; MicroRNA signature in wound healing following excimer laser ablation: Role of miR-133b on TGF- β, CTGF, collagen and smooth muscle actin synthesis in corneal fibroblast. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5223. doi: https://doi.org/.
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Stromal scarring is the result of a complex cascade of factors. The transforming growth factor-β (TGF-β system has been determined to play a key role in the formation of scar tissue. Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is a downstream mediator of many of the fibrotic actions of TGF- β. In this study, we determined the microRNAs (miRs) involved in corneal wound healing. We further examine the effect of miR-133b on the expression of CTGF, TGF-β, smooth muscle actin (SMA) and collagen rabbit corneal fibroblast (RbCF).
Laser ablated mouse corneas were collected at 0 and 2 days post ablation. RNA was collected from the corneas and samples were analyzed using the mouse cell differentiation & development miRNA PCR Array (QIAgen). The ability of miR-133b to target the 3’ UTR of TGF-β and CTGF was tested using a luciferase assay in RbCF. PCR was used to determine the effect of miR-133b on CTGF, TGF-β, SMA and collagen in RbCF. Finally, a migration assay was used to determine the effect of miR-133b on RbCF migration.
Two days after ablation, there were 37 out of 86 miRNAs with significant expression fold changes. Specifically, mir-133b had the greatest fold decrease at -14.33 and mir-22 had the greatest fold increase of 7.16. The 3’UTR region of CTGF was targeted by pre-miR-133b as indicated by a significant decrease of 38% (p<0.01) in the luciferase activity and there was no decrease in luciferase activity when miR133b was cotransfected with the plasmid expressing the 3’UTR region of TGF-β. When RbCF were stimulated with TGF-β, there is a significant decrease of miR-133b of 22% (p<0.01) whereas, there was a significant increase of CTGF and SMA of 31% and 80% (p<0.01), respectively. When RbCF were stimulated with TGF-β and pre-miR133b was added, there was a significant decrease in both CTGF and SMA of 30% and 37% (p<0.01) expression levels, respectively. And finally, there was a significant decrease in migration of RbCF when miR-133b was added compared negative control transfected RbCF.
These data indicate that miR-133b may play an important role in modulating profibrotic factors in corneal wound healing.
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