Circular RNA MELK Promotes Chondrocyte Apoptosis and Inhibits Autophagy in Osteoarthritis by Regulating MYD88/NF-κB Signaling Axis through MicroRNA-497-5p

Osteoarthritis (OA) is a rheumatic disease and its pathogenesis involves the dysregulation of noncoding RNAs. Therefore, the regulatory mechanism of circular RNA MELK (circMELK) was specified in this work. OA human cartilage tissue was collected, and circMELK, miR-497-5p, and myeloid differentiation factor 88 (MYD88) expression were examined. Human chondrocytes were stimulated with interleukin- (IL-) 1β and interfered with vectors altering circMELK, miR-497-5p, and MyD88 expression to observe their effects on cell viability, cell cycle and apoptosis, autophagy, and inflammation. The binding relationship between RNAs was verified. The data presented that OA cartilage tissues presented raised circMELK and MYD88 and inhibited miR-497-5p expression. IL-1β suppressed cell viability, prevented cell cycle, and induced apoptosis, autophagy, and inflammation of chondrocytes. Functionally, IL-1β-induced changes of chondrocytes could be attenuated by suppressing circMELK or overexpressing miR-497-5p. circMELK acted as a sponge of miR-497-5p while miR-497-5p was a regulator of MYD88. MYD88 restricted the effect of overexpressing miR-497-5p on IL-1β-stimulated chondrocytes. MYD88 triggered nuclear factor-kappaB (NF-κB) pathway activation. Shortly, CircMELK promotes chondrocyte apoptosis and inhibits autophagy in OA by regulating MYD88/NF-κB signaling axis through miR-497-5p. Our study proposes a new molecular mechanism for the development of OA.


Introduction
Osteoarthritis (OA) is a chronic degenerative joint disease and a major cause of pain and disability, characterized by chronic inflammation, progressive destruction of articular cartilage, and subchondral bone sclerosis [1]. e pathogenesis of OA involves mechanical, inflammatory, and metabolic factors that ultimately lead to the structural destruction and failure of synovial joints [2]. Chondrocyte apoptosis and autophagy are the most important pathological changes in OA [3]. erefore, exploring the regulatory mechanisms of chondrocyte apoptosis and autophagy may help to develop new strategies for OA treatment. Circular RNAs (circRNAs), specifically expressed in tissues and cells [4], are an important role in the progression of OA, such as circCDR1as [5], circPDE4D [6], and circRNA_Atp9b [7]. circRNAs exert their biological functions as ceRNAs of microRNAs (miRNAs) to regulate their downstream molecules [8]. A microarray analysis report reveals that hsa_circ_0009127 (circMELK) is one of the top 10 most upregulated circRNAs in postmenopausal osteoporosis patients, which may be involved in autophagy-related pathways, phosphatidylinositol 3-kinase-Akt signaling, forkhead box O signaling, and Ras-mitogen-activated protein kinase signaling [9].
Dysregulation of miRNAs is a key cause of a variety of human diseases, including OA [10,11], and is a critical role in chondrocyte development and cartilage homeostasis; miRNAs have also been shown to play a role in chondrocyte phenotype through regulating apoptosis, autophagy, and senescence, and regulation of miRNAs in joints is of utility to attenuate OA in animal models [12,13]. Based on these properties, miRNAs have been proposed as circulating biomarkers for OA. It has been confirmed that miR-497-5p expression is elevated in the serum of OA patients [14]. However, the role of miR-497-5p in OA remains controversial, because another study has described that miR-497-5p expression is decreased in OA cartilage and can attenuate IL-1β-induced degradation of chondrocyte cartilage matrix through Wnt/β-catenin signaling pathway [15].
We mainly explored the mechanism of circMELK in OA, as well as its interaction with miR-497-5p. Our cell experiments showed that circMELK promotes the apoptosis of OA chondrocytes and inhibits autophagy by regulating the myeloid differentiation factor 88 (MYD88)/nuclear factor-kappaB (NF-κB) signaling axis through miR-497-5p.

Results
We aimed to investigate the functional role of hsa_-circ_0009127 (MELK) in OA and the regulatory mechanism of hsa_circ_0009127 (MELK) regulating MYD88/NF-κB signaling pathway via miR-497-5p. We isolated and cultured human chondrocytes to construct an in vitro model and conducted a series of experiments. We found that hsa_-circ_0009127 (MELK) promotes OA chondrocyte apoptosis and inhibits autophagy by regulating the MYD88/NF-κB signaling axis through miR-497-5p. erefore, our data are the first to investigate the function and mechanism of hsa_circ_0009127 (MELK) regulating the MYD88/NF-κB signaling pathway through miR-497-5p in OA, providing new insights into the pathogenesis of OA.

circMELK Is Elevated in OA Cartilage
Tissue and IL-1β-Stimulated Chondrocytes. By analyzing circMELK expression in OA cartilage specimens and normal cartilage specimens, we found that circMELK expression was elevated in cartilage specimens tissues (Figure 1(a)). A cell model established by IL-1β stimulation was utilized to mimic OA in chondrocytes. After IL-1β stimulation, it was measured that the viability of chondrocytes was impaired (Figure 1(b)), G1 phase arrest was prevented (Figure 1(c)), and apoptosis was induced (Figure 1(d)). It has been accepted that OA progression is associated with insufficient autophagy in chondrocytes [28], and the activation of autophagy is often accompanied by increased expression of LC3 and beclin-1 [29]. Western blot analysis detected that IL-1β decreased beclin-1 expression (Figure 1(e)), GFP-LC3 immunofluorescence presented that IL-1β inhibited the number of GFP-LC3 (Figure 1(f )), and ELISA results showed that IL-1β raised the concentrations of proinflammatory cytokines, including IL-6 and TNF-α in chondrocytes (Figures 1(g) and   1(h)). Taken together, the OA cell model was successfully established. Notably, it was recognized that circMELK expression was elevated in IL-1β-treated chondrocytes (Figure 1(i)), implicating that circMELK may act as an inducer of OA.

An Interplay between circMELK and miR-497-5p.
Potential miRNAs regulated by circMELK were predicted from multiple databases to elucidate the action of circMELK in OA. Firstly, the characteristics of circMELK were analyzed. After RNase R treatment, MELK mRNA levels dropped, but circMELK exhibited strong resistance to RNase R digestion (Figure 3(a)). Under the action of actinomycin D, circMELK expression was more stable than linear MELK (Figure 3(b)). Nuclear isolation experiments showed that circMELK was enriched in the cytoplasm (Figure 3(c)). miR- 497-5p was identified by starbase as a downstream of circMELK for further study (Figure 3(d)). e luciferase reporter assay showed that when miR-497-5p was bound to circMELK-wt, the luciferase activity was reduced (Figure 3(e)). RNA pulldown identified the binding of circMELK to miR-497-5p, showing that miR-497-5p was pulled down by the biotinylated probe targeting circMELK (Figure 3(f )). In addition, miR-497-5p expression reduction was examined in both OA cartilage specimens and IL-1βtreated chondrocytes (Figures 3(g)) and 3(h)) and circMELK overexpression restricted miR-497-5p expression while circMELK downregulation induced miR-497-5p expression in IL-1β-treated chondrocytes (Figure 3(i)).

MYD88
Activates the NF-κB Pathway. By analyzing the activation of the NF-κB pathway, we further explored the molecular mechanism of the circMELK/miR-497-5p/ MYD88 axis in the progression of OA. As the data reported, MYD88 promoted the phosphorylation of IkBα, which facilitated the entry of p65 into the nucleus and ultimately activated the NF-κB signaling pathway (Figures 7(a) and 7(b)).

Discussion
Current treatments of OA can only relieve pain and cannot prevent cartilage damage and other joint tissue destruction [30]. e secretion of inflammatory factors has been reported in the pathological process of OA; therefore, IL-1βtreated human chondrocytes have been used to establish inflammatory injury models to study OA in vitro [31]. CircRNAs have recently been confirmed to be closely related to OA disease progression [32], based on which, we studied that circMELK is a novel OA causative factor and further testified that abnormal elevation of circMELK promoted OA chondrocyte apoptosis and inhibited autophagy by regulating the MYD88/NF-κB signaling axis through miR-497-5p.
Noncoding RNAs have been considered an effective way to reduce OA [33] and circRNAs function as functional RNAs, playing indispensably in OA [34]. It has been reported that articular cartilage degeneration is one of the important pathological processes of OA. Chondrocytes are the only resident cells in cartilage and maintain cartilage integrity. Chondrocyte apoptosis is an important factor in promoting OA [35]. In contrast, enhancement of chondrocyte autophagy prevents OA progression in articular cartilage [36]. Autophagy is an important protective mechanism that maintains the balance of anabolic and catabolic activities and maintains cellular homeostasis, and its dysfunction is a hallmark of OA pathogenesis [37]. erefore, inhibiting chondrocyte apoptosis and enhancing their autophagy are a promising therapeutic strategy for OA. Our study found that circMELK silence enhanced chondrocyte viability, accelerated cell cycle, and inhibited apoptosis, whereas circMELK overexpression did the opposite, suggesting that circMELK management might be a promising strategy for OA. It is well known that circRNAs are generally involved in the regulation of many diseases including OA by acting as miRNA sponges [38,39]. It has been documented that circMELK can act as a sponge for miRNAs, such as miR-593 [40], while in this work, miR-497-5p was an identified target of circMELK.
MYD88 promotes IL-1β-induced inflammation in human articular chondrocytes by activating the NF-κB signaling pathway [49]. NF-κB signaling pathway is involved in the regulation of various biological processes and closely related to the release of inflammatory cytokines [50,51]. erefore, we further explored whether miR-497-5p regulates OA through the MYD88/NF-κB axis. Indeed, we determined that miR-497-5p had an interplay with MYD88, detected that MYD88 was upregulated in OA chondrocytes, and confirmed that miR-497-5p inhibited MYD88 expression. Furthermore, we realized that MYD88 reversed the effect of overexpressing miR-497-5p on IL-1βstimulated chondrocytes by activating the NF-κB signaling pathway. It is undeniable that there are some limitations, mainly the insufficient sample size. In addition, further in vivo animal experiments are required to verify the effect of the circMELK/miR-497-5p/MYD88/NF-κB axis on OA progression. Finally, osteoblast dysfunction is also a key cause of OA [52], miR-497-5p has been shown to regulate osteoblast differentiation, and we hope to further explore the effect of circMELK on osteoblasts in OA in future studies to refine the molecular mechanism by which circMELK regulates OA progression.

Conclusion
All in all, circMELK regulates chondrocyte apoptosis and autophagy through the miR-497-5p-mediated MYD88/NF-κB signaling pathway, thereby promoting the progression of OA. is result helps to enhance the understanding of OA pathogenesis and provides new potential targets for OA prevention and treatment [53].

Data Availability
e figures used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Yingchi Zhang and Rui Lu contributed equally to this work and should be considered co-first authors.