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M. Chen, L. Wang, X. Wang, T. Watanabe, M. Kimura, T. Sakimoto, N. Fukuda, M. Sawa, H. Nagase; Pre-Transcriptional Regulation of Tgf-Beta1 and Mmp-9 by Novel Gene Silencers Accelerates Wound Healing of Alkali-Burned Cornea. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3098.
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Treatment of corneal alkali burns remains a major clinical challenge. Tgf-beta1 was up-regulated and involved in the response to corneal injury and the process of corneal stromal scarring. Over-expression of Mmp-9 exacerbates damage to injured cornea by persistent inflammation and degradation of the corneal epithelial basement membrane. PI polyamide has been proved to be a novel gene silencer for pre-transcriptional regulation of target gene expression. The aim of our study is to design PI polyamides targeting both genes, to evaluate the feasibility and therapeutic efficacy of polyamides in cornea alkali burn models.
PI polyamides were designed to target AP-1 binding site in the promoter region of the Tgf-beta1 and Mmp-9 genes. In vitro binding specificity and affinity to target DNA sequence was evaluated by DNA binding assays. Cornea alkali burns was produced in both eyes of rats and polyamides solution targeting Tgf-beta1 or Mmp-9 was applied to the right eye while the mock solution was applied to the left eye as a control. The subcellular localization of polyamides in alkali-burned eyes were examined by FITC-labelled polyamide. The time for cornea re-surface was recorded and the extent of corneal haze was evaluated. The mRNA and protein expression of Tgf-beta1, Mmp-9, Vegf and Mcp-1genes was evaluated by real-time PCR and Immunohistochemical analysis.
Polyamides showed high binding affinity and specificity to the target DNA.Topically administrated FITC-labelled polyamides were distributed immediately and sufficiently to the nuclei of all cell layers of cornea and remained for 24 hours without any delivery system. Tgf-beta1 targeting polyamide efficiently suppressed up-regulation of Tgf-beta1gene and its downstream genes Mmp-9, Vegf, Mcp-1, both in mRNA and protein levels, while Mmp-9 targeting polyamide presented specific regulation of the Mmp-9 gene. Wound healing of corneal tissues was remarkably improved in both groups although the Tgf-beta1 targeting polyamide contributed in a larger extent.
The favorable outcome could mean that pre-transcriptional Tgf-beta1 and Mmp-9 regulation by PI polyamide may open new opportunities for the treatment of corneal alkali burns.
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