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Eung Kweon Kim, Seung-il Choi, Kyu Seo Kim, Tae-im Kim, Hun Lee, Si Yoon Park; TGF-β regulate TGFBIp expression through coordination between miR-21 and miR-181a, and Smad signaling in corneal fibroblasts. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4829. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor-β (TGF-β)-induced gene (TGFBI) protein (TGFBIp) is associated with a granular corneal dystrophy type 2 (GCD2), and plays a role in tumorigenesis. Dosage of TGFBI can affect disease phenotypes of this disease, but the underlying molecular mechanisms have not been fully elucidated. We investigate here the contributions of microRNA (miRNA) and TGF-β to TGFBI expression in corneal fibroblasts.
Isolation, immortalization, and culture of primary corneal fibroblast were performed. RNA Isolation and Quantitative real-time PCR were done. Levels of miRNAs were normalized to RNU6B. Relative quantification was analyzed by the system software analysis based on 2−ΔΔCt method. The precursor miR-9, miR-21, miR-181a, miR-181a-3p, miR-181a-2-3p and negative miR precursor were transfected at a final concentration of 100 nM with G-Fectin and Lipofectamine™ 2000 reagent (Invitrogen Life Technologies). Western blotting was done with got anti-TGFBIp (R&D Systems, Minneapolis, MN). Statistical comparisons were performed using one-way analysis of variance (ANOVA) followed by Newman–Keuls multiple comparison tests.
Ectopic expression of miR-9, miR-21, and 181a reduce significantly TGBFIp levels. Further, the expressions of miR-21 and miR-181a were also induced by TGF-β1. The expression of miR-21 was 10-fold higher than miR-9 and miR-181a in corneal fibroblasts. Additionally, TGF-β1 expression was significantly higher than TGF-β2 and TGF-β3 in cornel fibroblasts, but levels of all three TGF-β forms are not significantly different between WT and GCD2 corneal fibroblasts. Taken together, these data indicated that TGFBIp expression are regulated by TGF-β positively and TGF-β-induced miR-21 and miR-181a negatively in corneal fibroblasts.
In conclusion, TGFBIp level in corneal fibroblasts is controlled through coordination between miR-21 and miR-181a, and Smad signaling. Pharmacologic modulation of these miRNAs and TGF-β may have therapeutic potential for TGFBI-linked corneal dystrophy including GCD2.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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