THU0508 CHANGES IN MIR-17–92 CLUSTER EXPRESSION LINK SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS, MONOCYTE-TO-MACROPHAGE DIFFERENTIATION, AND INTERFERON REGULATION
Authors: Divya Takkellapati, Xiaoling Niu, Thuy Do, Grant Schulert
Journal: Annals of the Rheumatic Diseases (2019)

<h3>Background:</h3> MicroRNAs (miRNAs) are small noncoding RNAs which post-transcriptionally regulate gene expression. The miR-17-92 cluster is well studied in cancer biology and cellular differentiation; its overexpression has been found to serve a major oncogenic role in the targeting and downregulation of tumor-suppressive pathways, such as PTEN or TGFß. Our previous work identified several members of the cluster – miR-18, miR-19a/b, miR-20a, and miR-92a – with significantly higher levels in monocytes from patients with active Systemic Juvenile Idiopathic Arthritis (SJIA). SJIA is a chronic inflammatory disease of childhood with features of autoinflammation, and innate immune cells including monocytes have important roles in disease pathogenesis. Children with SJIA are at risk for life-threatening complications including Macrophage Activation Syndrome (MAS), an episode of overwhelming inflammation characterized by macrophage proliferation and driven by IFNγ. <h3>Objectives:</h3> Characterize the regulation of the miR-17-92 cluster, define key targets, and determine the cluster's role in inflammation and SJIA. <h3>Methods:</h3> MiRNA levels were examined in THP-1 cells, as well as primary human monocytes isolated from healthy donors over the course of the monocyte to monocyte-derived macrophage (MDM) transition. MiR-17, miR-19a, and miR-20a were overexpressed via transfection in CD14+ monocytes for 2 days. Transcriptional profiles were performed using Ampliseq Transcriptome and the Ion Torrent S5 system and analyzed using AltAnalyze. Potential targets of the miR-17-92 cluster determined from sequencing analysis were then validated via dual-luciferase reporter assay. <h3>Results:</h3> Neither blood monocytes nor fully differentiated THP-1 cells showed significant changes in miR-17-92 levels under standard polarization conditions, including M1, M2a, and M2b conditions, or IL-6 and IL-10 stimulation. The most sizable changes in miR-17-92 levels were found during monocyte to macrophage transition. Interestingly, primary monocytes showed increases in miR-17-92 levels within the first 48 hours of differentiation towards MDM, variable by miRNA and experiment, similar to that seen in SJIA monocytes. In contrast, both PMA-differentiated THP1 cells and fully differentiated MDMs showed decreased miR-17-92 compared to undifferentiated monocytic cells. MiR-17-92 was overexpressed in vitro in primary monocytes to model these early transition changes. Genome-wide transcriptional profiling showed an upregulation of genes involved in Type I and II Interferon pathways, including response to interferon-alpha (adjusted p=2.71x10<sup>-12</sup>) and interferon-gamma (adjusted p=7.81x10<sup>-9</sup>). Analysis of genes significantly downregulated by miR-17, miR-19a, or miR-20a identified several putative and previously validated miR-17-92 cluster targets, including ATG5, IFRD2, JAK1, PPARG, and PTPN2 which have interferon-regulatory functions. Dual-luciferase reporter assay experiments support that these genes are direct targets of miR-17, miR-19a, and/or miR-20a. <h3>Conclusion:</h3> MiR-17-92 cluster members demonstrate initial increase followed by subsequent decrease in expression during 2-week human monocyte to macrophage differentiation. Overexpression of miR-17-92 miRNAs upregulates Type I and II interferon pathway genes, and these miRNAs target multiple genes involved in regulating interferon signaling and/or inflammatory response. Taken together, miR-17-92 cluster overexpression in SJIA monocytes may suggest a more differentiated phenotype, and contribute to IFNγ sensitivity and risk for MAS. <h3><b>Disclosure of Interests:</b>&nbsp;:</h3> None declared