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Tetsuya Toyono, Guadalupe Villarreal, Laura Kallay, Tomohiko Usui, Shiro Amano, Albert S Jun; MicroRNA29b over-expression decreases extra-cellular matrix protein secretion in Fuchs endothelial corneal dystrophy endothelial cells.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1175.
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MicroRNAs are small non-coding RNAs which regulate gene expression at the post-transcriptional level. We have reported that the microRNA (miR)-29 family which regulates extracellular matrix (ECM) expression is decreased in Fuchs endothelial corneal dystrophy (FECD) patient corneas compared to normal corneas. Accumulation of ECM proteins in Descemet membrane is an important pathologic change in FECD. In this study, we transfected miR29b into immortalized human corneal endothelial cells and evaluated ECM protein expression levels.
An immortalized FECD human corneal endothelial cell line was established by infection of an FECD patient’s corneal endothelial cells with hTERT lentivirus.MiR29b was transfected into human FECD corneal endothelial cells using RNAiMAX (Invitrogen) for 48 hours. Total RNA and protein were extracted. MiR29b expression change was evaluated with qPCR and ECM proteins (COL1A1, COL4A1, LAMC1) levels were evaluated with qPCR and Western blot.
Compared with control, miR29b expression level was increased to 335.6(±91.0)% and ECM protein expression levels were decreased. In qPCR results for miR29b transfected cells vs. controls, COL1A1 was 1.9(±1.4)%, COL4A1 was 7.2(±3.0)% and LAMC1 was 21.5(±7.0)%. In Western blot results for miR29b transfected cells vs. controls, COL4A1 was 42.5(±25.0)% and LAMC1 was 44.8(±3.1)%.
Over-expression of miR29b decreased ECM protein expression in FECD human corneal endothelial cells. Thus, miR29 replacement might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet membrane.
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