Thu0508 changes in Mir-17–92 Cluster Expression Link Systemic Juvenile Idiopathic Arthritis, Monocyte-to-Macrophage Differentiation, and Interferon Regulation

Abstract

Background: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.Objectives:Characterize the regulation of the miR-17-92 cluster, define key targets, and determine the cluster’s role in inflammation and SJIA.Methods: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.Results: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=2.71x10-12) and interferon-gamma (adjusted p=7.81x10-9). 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.Conclusion:MiR-17-92 cluster membe...