P. R. Varley1, Z. Guo1, D. A. Geller1 1University Of Pittsburgh,General Surgery,PITTSBURGH, PA, USA
Introduction: Redox stress is an important mediator of liver damage ischemia reperfusion (I/R) injury. Previously our group has described the role of inducible nitric oxide synthase (iNOS) in the liver, which exacerbates liver injury under redox stress. MicroRNA (miR)-155 is up-regulated by a number of inflammatory stimuli, however its role in the liver and effect on iNOS expression have not been reported. We hypothesized that miR-155 is induced in hepatocytes during inflammation and regulates iNOS expression.
Methods: Rat and human hepatocytes were isolated through collagenase dissociation and density gradient centrifugation. To determine the ability of miR-155 to regulate iNOS expression, primary human hepatocytes were isolated and treated with miR-155 mimics and then stimulated with a cytokine mix (CM) ((interferon-γ, IL-1β and TNF-α) for 24 hours. The Griess method was used to measure nitrite concentrations in the cell culture supernatant as endproduct for induced NO synthesis. To evaluate miR-155 expression, rat hepatocytes were treated with hydrogen peroxide (H2O2) and CM alone or in combination for up to 24 hours. Trizol purification was used to isolate RNA from the cells, and miR-155 levels were measured with TaqMan qRT-PCR.
Results: Transfection of primary human hepatocytes with miR-155 mimics resulted in near complete abrogation of nitrite production in CM-treated cells (NO2– 1.1 uM in miR-155+CM vs. 18.5 uM in CM alone, p < 0.05) suggesting significant suppression of cytokine-induced NO synthesis. H2O2 stimulation alone had a non-significant effect on miR-155 expression at 24 hrs, while CM stimulation resulted in significant up-regulation of miR-155 expression at 24hr (Table 1). Co-stimulation of hepatocytes with CM + H2O2resulted in a dramatic 21.3-fold increase in miR-155 expression at 24 hours.
Conclusion: This study is the first to describe down-regulation of iNOS by miR-155, a well-known inflammatory microRNA. Not only is miR-155 capable of iNOS regulation, but here we show that inflammatory cytokine stimuli under redox stress act in a synergistic fashion to induce miR-155. Our previous work has shown the iNOS expression peaks at 6-12 hours after introduction of inflammatory stimuli and returns to baseline by 24 hours. Taken together with the data from this study, we suggest that miR-155 blocks iNOS-mediated NO synthesis in a post-translational manner as a homeostatic control mechanism to prevent the untoward consequences of excessive iNOS expression and limit hepatocellular damage.
