Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


The microRNA 18a Enhances IL-6 Signaling through Inhibition of PIAS3.

Brock2,  Matthias, Trenkmann1,  Michelle, Gay3,  Renate E., Michel4,  Beat A., Gay5,  Steffen, Speich6,  Rudolf, Huber6,  Lars C.

Center for Experimental Rheumatology and Zurich Center for Integrative Human Physiology (ZIHP), University Zurich, Zurich, Switzerland
Center for Experimental Rheumatology and Zurich Center for Integrative Human Physiology (ZIHP), University Zurich, Zürich, Switzerland
Rheum Clinic, Univ Hospital, Zurich, Switzerland
University Hospital, Zurich, Switzerland
University Hospital Zurich, Zurich, Switzerland
Working Group for Pulmonary Hypertension, Department for Internal Medicine, University Hospital Zurich, Zurich, Switzerland

Introduction:

Interleukin-6 (IL-6), a key proinflammatory cytokine in the pathogenesis of certain diseases, mediates the hepatic production of cytokines and acute phase reactants such as fibrinogen gamma chain (FGG) and haptoglobin (Hp). We have shown that signal transducer and activator of transcription 3 (STAT3), which is the major intracellular mediator of IL-6 signaling, regulates the expression of the microRNA (miRNA) cluster miR-17/92. Here we addressed whether miRNAs derived from this cluster might modulate IL-6 signaling and acute phase response in human hepatoma (HepG2) cells.

Methods:

Overexpression of miRNA precursor molecules (pre-miR-17-5p, -18a, -19a, -20a and -92a) in HepG2 cells (n=6) was achieved by nucleofection (AMAXA). Following an incubation period of 72h, cells were stimulated with IL-6 for 4h. Gene expression of FGG, Hp, suppressor of cytokine signalling 3 (SOCS3), and protein inhibitor of activated STAT 3 (PIAS3) were measured by SYBR Green real-time PCR. HepG2 cells (n=5) were further transfected with reporter gene constructs of FGG or Hp promoters. 4h after stimulation with IL-6, cells were lysed and promoter activities were measured using the Dual-Luciferase Reporter Assay System (Promega).

Novel miRNA targets were identified by a computational algorithm (TargetScan) and validated by constructing a luciferase-based reporter gene system which contained the 3'UTR of the predicted target gene. Luciferase expression was assessed after co-transfection of HEK293 cells (n=7) with precursors of miR-18a and the respective reporter gene constructs.

Results:

Stimulation of HepG2 cells with IL-6 for 4h resulted in overexpression of mRNA levels of SOCS3 (48.11 ± 15.3), FGG (2.86 ± 0.93) and Hp (4.43 ± 1.0) as compared to unstimulated control cells. Additional transfection with miR-18a further increased these expression levels (SOCS3: 81.49 ± 20.84, p < 0.05; FGG: 3.89 ± 1.35, p = 0.08; and Hp: 6.31 ± 0.75, p < 0.05). These data could be confirmed by performing reporter gene assays revealing 15.96 ± 4.62 fold change in the promoter activity of FGG for mock transfected cells upon stimulation with IL-6 and 26.67 ± 4.15 fold change in pre-miR-18a transfected cells (p < 0.05). For Hp, similar results were achieved showing 15.48 ± 4.51 in mock and 26.4 ± 4.47 fold change in pre-miR-18a transfected cells (p < 0.05). Next, we identified PIAS3 as a novel target of miR-18a. Transfection of miR-18a resulted in reduced mRNA expression levels of PIAS3 (0.86 ± 0.09, p < 0.05). Similarly, the activity of Luciferase containing the 3'UTR of PIAS3 was reduced in pre-miR-18a transfected cells (0.127 ± 0.038) when compared to mock transfected cells (0.21 ± 0.044, p < 0.05). Conversely, this effect could be abrogated by mutation of the miR-18a seed match (0.247 ± 0.049 for mock and 0.265 ± 0.063 fold change for pre-miR-18a transfected cells).

Conclusion:

We show here that miR-18a enhances IL-6 signaling in HepG2 cells thereby increasing the production of acute phase proteins. We further identified PIAS3, an endogenous inhibitor of STAT3, as a direct target of miR-18a. Our data emphasize an important link between miR-18a and STAT3 activity in the regulation of inflammation and acute phase response.

To cite this abstract, please use the following information:
Brock, Matthias, Trenkmann, Michelle, Gay, Renate E., Michel, Beat A., Gay, Steffen, Speich, Rudolf, et al; The microRNA 18a Enhances IL-6 Signaling through Inhibition of PIAS3. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :35
DOI: 10.1002/art.27804

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