Proinflammatory cytokine interferon-γ increases the expression of BANCR, a long non-coding RNA, in retinal pigment epithelial cells

Abstract

The inflammatory response may contribute to retinal pigment epithelial (RPE) dysfunction associated with the pathogenesis of age-related macular degeneration (AMD). We investigated whether the inflammatory response affects the expression of long coding RNAs (lncRNAs) in human RPE-derived ARPE-19 cells. This class of regulatory RNA molecules recently came to prominence due to their involvement in many pathophysiological processes. A proinflammatory cytokine mixture consisting of IFN-γ, IL-1β and TNF-α altered the expression several lncRNAs including BANCR in these cells. The cytokine responsible for increasing BANCR expression in ARPE-19 cells was found to be IFN-γ. BANCR expression induced by IFN-γ was suppressed when STAT1 phosphorylation was blocked by JAK inhibitor 1. Thus, proinflammatory cytokines could modulate the expression of lncRNAs in RPE cells and IFN-γ could upregulate the expression of BANCR by activating JAK-STAT1 signaling pathway.

Keywords: Age-related macular degeneration; BRAF-activated non-coding RNA (BANCR); Interferon-γ; JAK-STAT1 signaling; Long non-coding RNA (lncRNA); Retinal pigment epithelium.

Fig. 1

Proinflammatory cytokines alter the expression of lncRNAs in RPE cells. Differentiated ARPE-19 cells were treated for 4 days with a proinflammatory cytokine mixture consisting of IFN-γ (100 u/ml), TNF-α (10 ng/ml) and IL-1β (10 ng/ml). (A), Phase contrast microscopic images of control and treated cells shows that proinflammatory cytokines caused a disruption in the epithelial morphology of ARPE-19 cells. Photomicrographs were taken at a magnification of 100X; scale bar = 100 μm. (B) Real-time PCR analysis shows that proinflammatory cytokines decrease the expression of RPE characteristic genes and miRNAs in ARPE-19 cells. * = p < 0.05 when compared to control, n = 3. (C) Real-time PCR analysis shows that proinflammatory cytokines alter the expression of lncRNAs in ARPE-19 cells. Relative expression level of lncRNAs affected by the treatment are shown (p < 0.05 when compared to control; n = 3). Broken line indicates that the relative expression level of an lncRNA in the control (untreated cells) is 1.

Fig. 2

IFN-γ regulates the expression of the lncRNA BANCR. (A) Proinflammatory cytokines increase the expression of BANCR in RPE cells. ARPE-19 cells were exposed to different concentrations of cytokine mixture for 4 days and BANCR expression analyzed by real-time PCR. 1X = IFN-γ (10 u/ml), TNF-α (1 ng/ml) and IL-1β (1 ng/ml). * = p < 0.05 when compared to control; n = 4. (B) IFN-γ by itself increases BANCR expression in RPE cells. The cells were treated with IFN-γ (100 u/ml), TNF-α (10 ng/ml) or IL-1β (10 ng/ml) for 4 days. A combination of all three cytokines was also used. BANCR expression was analyzed by real-time PCR. * = p < 0.05 when compared to control, n = 4. (C) JAK inhibitor 1 decreased BANCR expression induced by IFN-γ in RPE cells. ARPE-19 cells were treated with indicated concentration of IFN-γ for 20 hours with or without JAK inhibitor 1, and BANCR expression analyzed by real-time PCR. * = p < 0.05 when compared to control, ** = p < 0.05 when compared to - JAK inhibitor; n = 4. (D) JAK inhibitor decreased phospho-STAT1 (pSTAT1) content in ARPE-19 cells exposed to IFN-γ. The cells were treated with indicated concentration of IFN-γ for 20 hours in the presence or absence of JAK inhibitor 1, and the cell extracts were analyzed for pSTAT1 by western blotting. α-Tubulin was used as the gel loading control.