Interaction of RNA-binding protein HuR and miR-466i regulates GM-CSF expression

Jing Chen,William Adamiak,Abdolmohamad Rostami,Shiguang Yu,Ulus Atasoy,Ganlei Huang

doi:10.1038/s41598-017-17371-5

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T helper 17 (Th17) cells plays an essential role in autoimmune diseases. Transcriptional regulation of Th17 cell differentiation has been extensively studied, but post-transcriptional regulation of Th17 cell differentiation has remained less well characterized. The RNA-binding protein HuR functions to promote the stability of target mRNAs via binding the AU-rich elements of the 3′ untranslated region (3′UTR) of numerous pro-inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, whether HuR regulates GM-CSF expression in Th17 cells has not been fully investigated. Here we showed that HuR conditional knockout (KO) Th17 cells have decreased GM-CSF mRNA in comparison with wild-type (WT) Th17 cells, and that HuR binds directly to GM-CSF mRNA 3′UTR. Interestingly, HuR deficiency increased the levels of certain microRNA expression in Th17 cells; for example, miR-466i functioned to mediate GM-CSF and IL-17 mRNA decay, which was confirmed by in vitro luciferase assay. Furthermore, we found that HuR promoted Mxi1 expression to inhibit certain miRNA expression. Taken together, these findings indicate that interaction of HuR and miR-466i orchestrates GM-CSF expression in Th17 cells.

Highlights

Gene expression is post-transcriptionally regulated by RNA-binding proteins (RBPs)[23,24]
Considering that Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a non-redundant role in T helper 17 (Th17) cell induction of autoimmune demyelination[7,14], we were interested in determining if HuR regulates GM-CSF mRNA expression in Th17 cells
Quantitative real-time PCR data showed that KO Th0 and Th17 cells had significantly less GM-CSF mRNA as compared with WT counterparts on day 4 of culture (Fig. 1a,b)

Summary

Introduction

Gene expression is post-transcriptionally regulated by RNA-binding proteins (RBPs)[23,24]. Most RBPs destabilize target mRNAs, a few of these, including HuR, bind to 3′UTR of proinflammatory cytokines to stabilize them[24,25,26]. We previously showed that HuR post-transcriptionally stabilizes IL-17 and CCR6 mRNA and promotes their expression in autoimmune neuroinflammation[29,30]. It remains unclear whether HuR modulates GM-CSF expression in Th17 cells. MiRNAs are small, non-coding RNAs with approximately 21 to 24 nucleotides (nt)[33], which regulate the expression of numerous target genes by mediating their mRNA decay and/or repressing their translation[33,34,35]. In vitro luciferase transfection assay demonstrated that miR-466i could target GM-CSF and IL-17 mRNA 3′UTRs for decay, suggesting that miR-466i has potential as a novel reagent for therapeutic intervention in autoimmune inflammation

Methods

Results

Conclusion