Inhibition of miR-665 alleviates lipopolysaccharide-induced inflammation via up-regulation of SOCS7 in chondrogenic ATDC5 cells

Purpose: To examine the effect and mechanism of action of miR-665 in osteoarthritis. Methods: An in vitro inflammatory injury model of osteoarthritis was established using chondrogenic ATDC5 cells with lipopolysaccharide (LPS) treatment. The expression levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assays (ELISAs) and by quantitative real-time polymerase chain reaction (qRT-PCR). A binding target for miR-665 was predicted using TargetScan and then evaluated using a dual-luciferase reporter assay. Results: Treatment with LPS significantly up-regulated the inflammatory cytokine expressions of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α), in ATDC5 cells (p < 0.01), and the expression of miRNA-665 was significantly increased in LPS-treated ATDC5 cells (p < 0.01). Knockdown of miR-665 down-regulated the expression levels of these inflammatory cytokines. Suppressor of cytokine signaling 7 (SOCS7) was identified as a target of miR-665. Data from qRT-PCR and western-blot analyses indicated that SOCS7 expression was promoted by miR-665 inhibition and inhibited by miR-665 over-expression. LPS treatment significantly decreased the expression of SOCS7 protein in ATDC5 cells (p < 0.01), and over-expression of SOCS7 attenuated the LPS-stimulated inflammatory injury. In addition, over-expression of miR-655 enhanced the inflammatory injury and reversed the protective effect of SOCS7 against LPS-stimulated inflammation. Conclusion: Inhibition of miR-665 alleviated LPS-stimulated inflammatory injury in ATDC5 cells via the up-regulation of SOCS7, suggesting a potential therapeutic target for osteoarthritis.


INTRODUCTION
The clinical manifestations of osteoarthritis, including recurrent joint pain and the progressive worsening of joint-movement disorders, has a large impact on the health of the elderly around the world [1]. Chondrocytes are regarded as the main biomechanical elements leading to osteoarthritis [2], and osteoarthritis is strongly affected by chondrocyte inflammation [2]. Although joint-replacement therapy may be recommended at later stages of osteoarthritis [2], new therapeutic targets are urgently needed to inhibit chondrocyte inflammation. Research has indicated that microRNAs (miRNAs) play a critical role in the regulation of osteogenesis, and affect the catabolism and anabolism of bone and cartilage [3]. A newly discovered inflammatory regulator, miRNA-665, has been shown to reduce inflammation and apoptosis during intestinal ischemia/reperfusion [4] and is now known to be associated with osteogenic differentiation of adipogenic stem cells [5]. However, its effects on inflammation in osteoarthritis have yet to be reported. Suppressor of cytokine signaling (SOCS) proteins, significant physiological regulators of cytokine responses, have demonstrated significant implications for osteoarthritis pathology [6]. For example, SOCS1 has been shown to negatively regulate the inflammatory response in osteoarthritis cartilage [7]. However, whether SOCS proteins are involved in miR-665 regulation of osteoarthritis has not been determined.
In the present study, we first determined the expression level of miR-665 in lipopolysaccharide-(LPS) induced chondrogenic ATDC5 cells, and then investigated both the effect of miR-665 on inflammation in ATDC5 cells and the underlying mechanism. The results suggest that miR-665 is a potential therapeutic target for osteoarthritis.

Assessment of inflammatory cytokines
ELISA kits (R & D Systems, Minneapolis, MN, USA) were used to detect levels of IL-1β, IL-6 and TNF-α in the culture supernatant of ATDC5.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA or miRNAs from ATDC5 cells were isolated and then reverse-transcribed into cDNA. SYBR Green Master Mix (Roche, Mannheim, Germany) was used for the qRT-PCR analyses with endogenous controls (GAPDH or U6). The primer sequences are shown in Table 1.

ID
Sequence

Dual-luciferase reporter assay
Both wild type and mutant SOCS7 sequences were subcloned into the pmirGLO luciferasereporter vector (Promega, Madison, Wisconsin, USA).
Chondrogenic ATDC5 cells were seeded (3 × 10 4 /well), and then co-transfected with the luciferase-reporter vectors along with miR-665 inhibitor, or negative control. After 48 h, luciferase activity was assayed using the Lucifer Reporter Assay System (Promega).

Statistical analysis
All results were expressed as means ± SEMs. Using GraphPad Prism 7.0 software, the data were evaluated using a one-way analysis of variance. p < 0.05 was considered statistically significant.

LPS-induced inflammatory response, and upregulation of miR-665 expression in ATDC5 cells
Data from the qRT-PCR analyses (Figure 1 A, left panel) indicated that LPS treatment (10 μg/ml) significantly up-regulated the mRNA expression levels of pro-inflammatory factors (IL-1β, IL-6, and TNF-α) in ATDC5 cells. This LPSinduced inflammatory response in ATDC5 cells was also confirmed by ELISA, as demonstrated by the up-regulation of pro-inflammatory factors (Figure 1 A, right panel). Furthermore, miR-665 was up-regulated in LPS-stimulated ATDC5 cells (Figure 1 B), suggesting a possible regulatory role for miR-665 in osteoarthritis.

Inhibition by miR-665 alleviated the LPSstimulated inflammatory response
First, the transfection efficiency of the miR-665 inhibitor was confirmed (Figure 2 A), and then both the qRT-PCR and ELISA analyses indicated that the miR-665 inhibitor dramatically downregulated the pro-inflammatory factors in LPSinduced ATDC5 cells (Figure 2 B).

SOCS7 as a binding target for miR-665
Using a TargetScan analysis, SOCS7 was identified as a potential binding target for miR-665 (Figure 3 A). Application of the miR-665 inhibitor decreased the luciferase activity of the wild type SOCS7 reporter vector, while the same inhibitor had no significant effect on the mutant SOCS7 reporter (Figure 3

Over-expression of SOCS7 alleviated the LPS-stimulated inflammatory response
In ATDC5 cells, LPS induced a significant decrease of SOCS7 (Figure 4 A), while over-expression of SOCS7 reversed this LPS-induced SOCS7 suppression (Figure 4 A). In addition, over-expression of SOCS7 reduced the LPSstimulated increase in mRNA and protein levels of pro-inflammatory factors (Figure 4 B).

DISCUSSION
To date, 16 miRNAs have been found to be dysregulated in patients with osteoarthritis compared to people with normal cartilage [8].
Here, we evaluated the effect and underlying mechanism of miR-665 in a model of osteoarthritis.
Although osteoarthritis has traditionally been regarded as a non-inflammatory disease, inflammatory responses have been shown to contribute to its pathogenesis [9]. Inflammatory responses in articular cartilage promote the secretion of pro-inflammatory factors from chondrocytes, and result in the degeneration of articular cartilage and the development of osteoarthritis [10].
Lipopolysaccharide is known, both in vitro and in vivo, to stimulate inflammatory responses via increases in pro-inflammatory factors [11]. In addition, the chondrogenic cell line, ATDC5, has long been used as an in vitro model for chondrogenesis [12]. Recently, an LPSstimulated chondrocyte model was developed for investigating the pathogenesis of osteoarthritis [13]. In the present study, LPS-treated ATDC5 cells have been established as a model for testing new drugs against osteoarthritis.
Previous research has shown that LPS treatment can promote the secretion of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α [11].
Here, we demonstrate that the expressions of pro-inflammatory factors were also increased in LPS-treated ATDC5 cells, suggesting that the treatment represents an inflammatory injury to these cells. IL-1β functions as an important regulator of the inflammatory response, promoting chondrocyte apoptosis, and articular cartilage damage during the development of osteoarthritis [14].
Interleukin 6 mediates the proliferation and activation of T-and B-cells in the inflammatory process [15], and tumor necrosis factor-α also acts as a trigger for the inflammatory response [11]. Inhibition of these pro-inflammatory factors has been shown to be promising for ameliorating inflammatory injuries in osteoarthritis [11]. Although we have demonstrated an antiinflammatory effect of miR-665 inhibition in LPStreated ATDC5 cells, an underlying mechanism is still unclear. We identified SOCS7 as a binding target for miR-665 in this study, and as a member of the SOCS protein family regulating cytokine responses, SOCS7 has been implicated in allergic inflammation of mast cells via thymic stromal lymphopoietin [16], in insulin resistance [17], and over-expression of SOCS7 attenuated the inflammation in mice with unilateral urethral obstruction [18]. This study has shown that LPS stimulated a decrease in SOCS7, and that overexpression of SOCS7 relieved inflammation by decreasing the expression of pro-inflammatory factors.
Moreover, the anti-inflammatory effect of SOCS7 was reversed by an miR-665 mimic, revealing the critical role of the miR-665/SOCS7 axis for mediating LPS-stimulated inflammatory injury in ATDC5 cells. However, a previous study has shown that nuclear factor-κB, SRY-related highmobility-group box 9-related, and other signaling pathways are also associated with miRNA regulation in osteoarthritis pathogenesis [19], so the signaling pathways associated with regulation of the miR-665/SOCS7 axis in osteoarthritis also need further investigation.

CONCLUSION
MicroRNA-665 exhibits a pronounced proinflammatory effect in LPS-induced inflammation in ATDC5 cells; inhibition of miR-665 is manifested as an anti-inflammatory effect in this model of osteoarthritis via regulation of SOCS7. These findings reveal that the miR-665/SOCS7 axis may be a new potential therapeutic target for osteoarthritis.