Elsevier

Biomedicine & Pharmacotherapy

Volume 96, December 2017, Pages 208-214
Biomedicine & Pharmacotherapy

Original article
MiR-140-5p inhibits synovial fibroblasts proliferation and inflammatory cytokines secretion through targeting TLR4

https://doi.org/10.1016/j.biopha.2017.09.079Get rights and content

Abstract

Objectives

This study was aimed to investigate the effects of miR-140-5p on the proliferation and inflammatory cytokines secretion of rheumatoid arthritis synovial fibroblasts (RASFs).

Methods

Synovial tissue samples from 23 rheumatoid arthritis (RA) patients and 18 normal synovial tissue samples were collected. The RASFs were isolated and cultured. Then, miR-140-5p and TLR4 expression in both synovial tissue and RASFs were detected using the quantitative real-time PCR (qPCR) and western blot. Dual luciferase reporter gene assay was employed to evaluate the interaction between miR-140-5p and 3′UTR of TLR4. Western blotting and qPCR were used to examine TLR2 expression after upregulation or downregulation of miR-140-5p in RASFs. After RASFs co-infected with TLR4 overexpression lentivirus and lentivirus containing miR-140-5p or miR-control respectively, the cellular proliferation and secretion of IL-6 and IL-8 level were detected through the MTS assay and enzyme-linked immunosorbent assay, respectively.

Results

MiR-140-5p was significantly down-regulated, and TLR4 was significantly up-regulated in synovial tissue samples from 23 RA patients and RASFs. Dual luciferase activity assay showed that miR-140-5p could specifically bind to the 3′UTR of TLR4. Down-regulation or up-regulation of miR-140-5p not only significantly increased or decreased the expression of TLR4, but also could promote or inhibit RASF proliferation and secretion of IL-6, and IL-8 in RASFs. Furthermore, overexpression of TLR4 can reverse the inhibitory effects of miR-140-5p on proliferation and inflammatory cytokines release of RASFs.

Conclusions

MiR-140-5p could inhibit the proliferation and secretion of IL-6 and IL-8 through regulation of TLR4 expression.

Introduction

Rheumatoid arthritis (RA) is a systemic chronic autoimmune disease that is characterized by pronounced synovial hyperplasia, joint synovitis as well as the bone and cartilage destroyed induced by inflammatory cytokines and proteases [1], [2], [3]. The morbidity of RA is approximately 5–50 per 100,000 populations, and 0.5–1% of adults are affected by RA in developing and developed countries [4]. It is considered that RA is caused by the interaction of immunological, genetic and environmental factors [5]. In addition, many studies have reported that proliferation and cytokine release of fibroblast-like synoviocytes (FLS) play a key role in the occurrence and progression of RA [6], [7], [8]. In this study, the elements that affect synovial fibroblasts activities were investigated.

Toll-like receptors (TLRs) are pattern recognition receptors that regulate innate immunity and defend invading microorganisms [9]. Besides, TLRs also mediate autoimmunity. Upon stimulation, TLRs initiate the downstream signaling pathways and lead to the secretion of inflammatory chemokines and cytokines [10]. TLRs are involved in the regulation of the pathogenesis of RA. Several studies have shown that the expression of TLR2 and TLR4 is increased in macrophages and synovial fibroblasts of RA patients, compared with healthy control or osteoarthritis (OA) patients [11], [12]. Lipopolysaccharides (LPS) treatment induces the high expression of TLR4 in peripheral blood mononuclear cells from RA patients, and then leads to dramatically increase of IL-6 [13]. It has been reported that IL1rn−/− TLR4−/− mice decrease the number of Th17 cell and the release of IL-17, then show a protective effect on severe arthritis [14]. These results suggested TLR4 might be a therapeutic target of RA.

miRNAs is a class of endogenous ∼22 nucleotide (nt) single-strand and highly conserved noncoding RNAs, which are predicted to regulate about 30% of gene expression via interfering mRNA translation [15]. It has been reported that miRNAs play a multifunctional role in many physiological and pathological processes including the development of RA [16]. For instance, a recent study has shown that miR-650 inhibits proliferation, migration and invasion of RASFs by targeting AKT2 [17]. Moreover, another report has indicated that miR-19 contributes to RA progression by suppressing FLSs cytokine release via targeting TLR2 [18]. These studies make miRNAs potential targets in RA treatment.

These above studies prompted us to discovery miRNAs directly targeting TLR4 and regulating inflammation in RA. Here, we found that miR-140-5p was down-regulated in RA patients, could directly bind to the 3′UTR of TLR4 and downregulate its expression. Furthermore, our results showed that miR-140-5p inhibited synovial fibroblasts proliferation and inflammatory cytokines secretion through targeting TLR4, which suggest that recovering the expression of miR-140-5p might be usefully therapeutic treatment for RA.

Section snippets

Object of study

Synovial tissue samples from 23 RA patients (14 females and 9 males, 27–73 years old, mean 55 years old) were collected during joint surgery at The Third Hospital of Hebei Medical University from 2010 to 2015. All RA patients were diagnosed according to the American College of Rheumatology criteria for classification of disease [19]. Healthy control specimens (10 females and 8 males, 30–69 years old, mean 52 years old) were collected from joint trauma patients undergoing joint replacement

Expression of miR-140-5p in synovial tissues and RASFs

To study the role of miR-140-5p in rheumatoid arthritis, we detected the expression of miR-140-5p in synovial tissue samples from 23 RA patients and 18 healthy controls. The results showed that miR-140-5p was significantly down-regulated in RA patient compared with healthy control (P < 0.001, Fig. 1A). In addition, the expression of miR-140-5p in HSFs and RASFs was tested. Compared with HSFs, the expression of miR-140-5p was significantly decreased in RASFs (P < 0.001, Fig. 1B). These results

Discussion

Emerging data have demonstrated that miRNAs are closely related to the pathological progression of RA [16]. It has been reported that numerous miRNAs such as miR-19 [18], miR-451[25], miR-537 [26], miR-203 [27], miR-17 [28] and miR-650 [17] are involved into the regulation of RA pathogenesis. Thus, miRNAs also have been believed as therapeutic strategy for RA treatment [16]. For instance, intraarticular delivery of miRNA-140-5p can ameliorate autoimmune arthritis [29]. However, the mechanism of

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work didn’t receive any funding support.

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