PDF《International Immunopharmacology》

miR-709 modulates LPS-induced in?ammatory response through targeting GSK-3β Ming Li, Hu Chen, Luxi Chen, Yaosheng Chen, Xiaohong Liu, Delin Mo ? State Key laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, PR China a b s t r a c t a r t i c l e i n f o Article history: Received

27 January

2016 Received in revised form

18 March

2016 Accepted

4 April

2016 Available online

25 May

2016 MicroRNAs (miRNAs) are endogenous small non-coding RNAs which modulate gene expression at the post- transcriptional level by either translational inhibition or mRNA degradation.

MicroRNAs play important roles in both innate and adaptive immune response, including TLR-triggered immune response. In this study, we found that the expression of miR-709 was up-regulated in primary macrophage and RAW264.7 cells during the stimulation of LPS. Overexpression of miR-709 in RAW264.7 cells led to reduced production and gene expres- sion of in?ammatory cytokines (IL-6, TNF-α, IL-1β) during activation by LPS, whereas knockdown of miR-709 had completely opposite effects. We used bioinformatics and experimental techniques to demonstrate that GSK-3β is a direct target of miR-709. miR-709 mimics decreased GSK-3β protein but not mRNA level. We also found that miR-709 regulated the LPS-induced in?ammatory response by targeting GSK-3β and elevating β-catenin. In conclusion, our data revealed a novel role for miR-709 in regulation of in?ammatory response by targeting GSK-3β. ?

2016 Published by Elsevier B.V. Keywords: miR-709 In?ammatory response GSK-3β β-Catenin 1. Introduction The mammalian in?ammatory response is a rapid biological reaction to invading pathogens including microbial products. Robust activation of the in?ammation is critical for host defense [1C4]. However, if it was not regulated properly, the response can be harmful to the host, leading to a variety of pathologies, ranging from chronic in?ammation, autoimmunity disease and cancer [5]. Thus, it is not surprising that a dis- tinct group of regulators including a complicated network of transcrip- tional factors and receptors are involved in the modulation of in?ammation. In recent years, although much of the focus has been on the mechanisms of in?ammatory response, it remains unclear how this complicated system was regulated. MicroRNAs are highly conserved noncoding RNA which were found to be important regulators of gene expression in the immune responses. They induce gene degradation or translational suppression through base pairing to the 3′-untranslated region of targeted mRNA [6]. A growing body of evidence indicates that the initiation and development of immune response is particularly subject to regulation by miRNA [7]. In our previous study, we measured the expression level changes of miRNA using high-throughput miRNA microarray technology in mouse spleens after LPS injection [8]. Nineteen miRNAs were found to be up- regulated at least 1.5 folds in mouse spleen tissue post LPS treatment. Among them, miR-709 was up-regulated N2 folds upon LPS challenge compared with saline treatment. However, the mechanism by which miR-709 modulates in?ammatory responses are still unclear. In this study, we identi?ed a novel mechanism of in?ammatory response mod- ulation by miR-709 via Wnt/β-catenin pathway. 2. Materials and methods 2.1. Reagents Lipopolysaccharide was purchased from Sigma. miR-709 mimics and negative control, miR-709 inhibitor and inhibitor control, GSK-3β small interfering RNA (siRNA) and control siRNA were from Ruibo Bio- technology (Guangzhou, China). 2.2. Cell culture and transfection RAW264.7 cells and human embryonic kidney (HEK) 293T cells were purchased from American Type Culture Collection and maintained in DMEM supplemented with 10% FBS and antibiotics (100 U/ml peni- cillin and