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R K. Kutty, C N. Nagineni, W. Samuel, C. Vijayasarathy, C. Jaworski, T. Duncan, J J. Hooks, T M. Redmond; Inflammatory Cytokines Differentially Regulate the Expression of MicroRNA-146a and MicroRNA-146b in Human Retinal Pigment Epithelial (RPE) Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2563.
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The inflammatory response of RPE is implicated in the pathogenesis of age-related macular degeneration (AMD). The microRNAs miR-146a and miR-146b are known to regulate the inflammatory process by their ability to target IRAK1 and TRAF6, two important genes involved in cytokine signaling, for translational repression. The aim of the present study is to investigate the expression of miR-146a and miR-146b in human RPE cells and their response to inflammatory cytokines.
Confluent cultures of RPE cells established from human donor eyes were treated with IFN-γ, TNF-α and IL-1β either individually or in combination in a serum free medium for 16 hours. Total RNA fraction containing miRNA was isolated using mirVana miRNA isolation kit (Applied Biosystems). The expression of miR-146a and miR-146b was analyzed by real-time PCR using TaqMan reagents (Applied Biosystems).
Real-time PCR analysis showed that miR-146a and 146b are expressed in RPE cells. Treatment of these cells with an inflammatory cytokine mix (IFN-γ + TNF-α + IL-1β) resulted in an increase in expression of miR-146a by ~35-fold and that of miR-146b by ~6-fold. The miR-146a induction was dependent on IL-1β since its omission from the cytokine mix abolished the response. Similarly, the induction of miR-146b was dependent on IFN-γ since its omission from the cytokine mix prevented this effect. The induction of both miR-146a and miR-146b by the cytokine mix was blocked by JAK inhibitor 1, a known inhibitor of JAK/STAT signaling pathway. However, miR-146b exhibited higher sensitivity to this inhibition. The increase in miR-146a and miR-146b expression in RPE cells in response to cytokine mix was also associated with marked increases in the expression of CCL2, CCL5, CXCL9, CXCL10 and IL-6 transcripts as well as their corresponding proteins.
Our results clearly show that both miR-146a and miR-146b are expressed in human RPE cells and their expression is highly increased by the inflammatory cytokine mix (IFN-γ, TNF-α and IL-1β). The induction of miR-146a showed a dependency on IL-1β, whereas that of miR-146b on IFN-γ. These two microRNAs could be important regulators of inflammatory processes underlying AMD or other retinal degenerative diseases.
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