MyD88 as a target of microRNA-203 in regulation of lipopolysaccharide or Bacille Calmette-Guerin induced inflammatory response of macrophage RAW264.7 cells

Highlights

• miR-203 was directly targeting the 3′ untranslated region (3′UTR) of MyD88 and down-regulating the expression of protein.

• Overexpression of miR-203 was correlated with repressions of MyD88, as well as NF-κB, TNF-α and IL-6.

• miR-203 may be an important regulator in macrophages against LPS or mycobacteria infection.

Abstract

MicroRNAs (miRNAs) have been demonstrated to play a pivotal role in the regulation of target gene expression at the post-transcriptional level. In order to better understand the role of microRNA-203 (miR-203) in the immunological regulation, the function of miR-203 was explored in the macrophage RAW264.7 cells against lipopolysaccharide (LPS) or Bacille Calmette-Guerin (BCG) stimulation. The results evidenced that myeloid differentiation factor 88 (MyD88) was a novel target of miR-203, miR-203 was capable of directly targeting the 3′ untranslated region (3′UTR) of MyD88 and post-transcriptionally down-regulating the expression of protein. In addition, an overexpression of miR-203 in RAW264.7 cells was correlated with repressions of MyD88, as well as its downstream signaling of NF-κB (NF-κB1), TNF-α and IL-6. These results suggest that miR-203 may be an important regulator in macrophages against LPS or mycobacteria infection, which may through a mechanism of negatively regulating MyD88-dependent Toll-like receptors signaling pathway.

Introduction

The innate immune system is activated through recognition of pathogen-associated molecular patterns (PAMPs) by toll-like receptors (TLRs). After recognizing a PAMP, TLRs bind adaptor proteins, such as MyD88 (most TLRs, with the exception of TLR3, use a MyD88-dependent pathway), which in turn, recruits the IRAK complex, including four subunits: two active kinases (IRAK-1 and IRAK-4) and two noncatalytic subunits (IRAK-2 and IRAKM) (O‘Neill and Bowie, 2007, Takeda et al., 2003). Subsequently, IRAK-1 is phosphorylated by IRAK-4, and the phosphorylated IRAK-1 associates with TRAF6 to activate signaling of the NF-κB and MAPKs (Lang et al., 2003, Moynagh, 2009, Rakoff-Nahoum and Medzhitov, 2009). In the case of NF-κB activation, the IRAK-TRAF6 complex leads to phosphorylation and degradation of IκB via the IKK complex, enabling the nuclear translocation of NF-κB. The activation of NF-κB and MAPK induces the transcription of various inflammatory genes, including IL-1β, IL-6, and TNF-α (Lang et al., 2003, Rakoff-Nahoum and Medzhitov, 2009), leading to proinflammatory or innate immune responses.

MicroRNAs (miRNAs) is a sort of endogenous noncoding small RNAs of ~22 nucleotides, which have been demonstrated to play a crucial role in the regulation of gene expression at the posttranscriptional level. Partial complementary pairing structure of miRNAs is able to bind the region of 3′UTR of target mRNA, and thus the silence the expression of target gene (Bartel, 2004). It is believed that miRNAs involved in the regulation of up to 30% of human genes, some of which were involved in the modulation of innate immune responses (Rajewsky, 2006). In response to stimulation of LPS or other microbial components, a rapidly increased expressions of miRNAs let-7e (Androulidaki et al., 2009), miR-146 (Taganov et al., 2006), miR-21 (Sheedy and O‘Neill, 2008), miR-155 (O‘Connell et al., 2007), and/or miR-181c (O‘Connell et al., 2009), have been observed in monocytic cell lines or mouse macrophages. These miRNAs were able to modulate inflammatory responses by targeting different components of TLR signaling pathways, such as TLR4, IL-1R-associated kinase 1/TNFR-associated factor 6, PDCD4, SCOS1/SHP1, and TNFSF11 (RANKL). In addition, down-regulated expression of miRNAs was also observed in some circumstances. For instance, miR-125b was able to target TNF-α, its expression was suppressed following LPS stimulation (Tili et al., 2007). However, the underlying mechanisms of miRNAs in the regulation of immune responses against varied pathogens or stimuli remain poorly understood, which await further investigation. The aim of this study was to explore the role of miR-203 in modulation of inflammatory response against LPS or BCG stimulation in macrophage RAW264.7 cells.