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MicroRNA-128-3p suppresses interleukin-1β-stimulated cartilage degradation and chondrocyte apoptosis via targeting zinc finger E-box binding homeobox 1 in osteoarthritis

ABSTRACT Accumulating studies have suggested that microRNAs (miRNAs) play vital roles in the pathogenesis of osteoarthritis (OA). Nevertheless, the specific function of miR-128-3p in OA remains unknown. In this study, we demonstrated that miR-128-3p was decreased and ZEB1 was increased in OA. Additionally, miR-128-3p expression was negatively correlated with ZEB1. miR-128-3p overexpression or ZEB1 silencing attenuated extracellular matrix degradation and cell apoptosis, and increased the proliferation of IL-1β-activated CHON-001 cells. Furthermore, ZEB1 was directly targeted by miR-128-3p. In addition, ZEB1 upregulation restored the effects of miR-128-3p overexpression on OA progression. Overall, our findings suggested that miR-128-3p might regulate the development of OA via targeting ZEB1.


Introduction
Osteoarthritis (OA) is considered as the most common age-related chronic joint disease, which is associated with severe inflammation, cartilage destruction and joint degeneration [1][2][3]. OA may cause pain or even disability in some severe cases. Despite the efforts made to improve the therapeutic approaches to OA, the treatment outcomes remain unsatisfactory due to the complex pathogenesis of the disease [4][5][6]. Therefore, it is important to investigate the molecular mechanisms underlying OA.
It has been reported that ZEB1 is implicated in inflammatory response. For example, miR-708 inhibited liver inflammation and lipid accumulation via targeting ZEB1 [13]. In addition, ZEB1 promoted inflammation via suppressing the DNA repair glycosylase MPG in epithelial cells [14]. Lai et al demonstrated that the expression levels of ZEB1 in synovial fluid and chondrocytes were higher in knee OA compared to the control group, and ZEB1 was inversely correlated with miR-100-5p [15], suggesting that ZEB1 might act as a potential diagnostic biomarker in OA. Nevertheless, the effect of ZEB1 in OA remains elusive.
The present study was undertaken to determine the levels of miR-128-3p and ZEB1 in OA and explore the role of miR-128-3p in modulating cartilage ECM degradation, cell viability and apoptosis of IL-1β-stimulated chondrocytes. The aim of this study was to provide new insights into the treatment of OA.

Cell culture and treatment
The chondrogenic cell line (CHON-001) was purchased from ATCC and grown in DMEM with 10% FBS at 37°C with 5% CO 2 . Then, CHON-001 cells were stimulated with 10 ng/ml IL-1β (Sigma-Aldrich) for 24 h to generate an in vitro OA model as previously described [16,17].

CCK-8 assay
CHON-001 cells (5x10 3 ) were seeded into 96-well plates and incubated for 24, 48 and 72 h. Then, 10 μl of CCK-8 solution was added to each well, and the cells were incubated for an another 2 h. The absorbance was measured using a microplate reader (Bio-Rad Laboratories, Inc.) at 450 nm [19].

TUNEL assay
The One Step TUNEL Apoptosis Assay Kit (Beyotime) was utilized to determine CHON-001 cell apoptosis [20]. Following dehydration in ethanol, cells were dyed and cultured with a TUNEL reaction mixture (Roche Diagnostics). Nuclear staining was performed with DAPI. TUNELpositive cells were observed under a fluorescence microscope (Nikon Corporation).

Luciferase reporter assay
The plasmids containing the wild-type (WT) and mutant (MUT) ZEB1 3′UTR were synthesized by GenePharma Co., Ltd (Shanghai). Subsequently, these plasmids were co-transfected with miR-128-3p or NC mimics into cells. After 48 h, the luciferase activity was detected using the Dual-Luciferase Reporter Assay system (Promega Corporation) [21].

Immunoprecipitation (RIP) assay
RIP assay was conducted by Magna RNA-binding protein RIP kit (EMD Millipore) [22]. CHON-001 cells were lysed with RIP lysis buffer and incubated with magnetic beads conjugated with Ago2 or IgG. The RNAs in the immunoprecipitates were extracted with Trizol reagent and detected by RT-qPCR.

Statistical analysis
The data are expressed as the mean ± SD. Statistical analysis was performed using the SPSS software (IBM.). Comparisons between two groups were carried out by Student′s t-test, while those among multiple groups with one-way ANOVA. P < 0.05 was considered to indicate a statistically significant difference.

Results
The aim of this study was to investigate the role of miR-128-3p in modulating cartilage ECM degradation, cell viability and apoptosis of IL-1β-stimulated chondrocytes. Through a series of functional experiments, we demonstrated that miR-128-3p suppresses IL-1β-stimulated cartilage degradation and chondrocyte apoptosis via targeting ZEB1 in osteoarthritis.

miR-128-3p is downregulated and ZEB1 is upregulated in OA
To determine whether miR-128-3p and ZEB1 were dysregulated in OA patients, the expressions of miR-128-3p and ZEB1 were measured in OA cartilage tissues. The results demonstrated that the expression of miR-128-3p was reduced and that of ZEB1 was enhanced in OA cartilage tissues (Figure 1(a,b)). In addition, a negative correlation was observed between miR-128-3p and ZEB1 expression in OA tissues (Figure 1(c)). Furthermore, CHON-001 cells were treated with IL-1β to establish in vitro OA model. As shown in Figure 1(d), RT-qPCR revealed that miR-128-3p expression was notably reduced in IL-1βtreated cells. In addition, ZEB1 expression was strongly upregulated following cell treatment with IL-1β (Figure 1(e)). Overall, the aforementioned findings suggested that the abnormal levels of miR-128-3p and ZEB1 might be involved in the progression of OA.

ZEB1 silencing ameliorates the effect of IL-1β on chondrocytes
Next, the effects of ZEB1 on OA development were evaluated. RT-qPCR assay showed that ZEB1 expression was reduced by ZEB1 silence (Figure 3(a)). Meanwhile, ZEB1 knockdown restored the IL-1βmediated upregulation of ZEB1 in CHON-001 cells (Figure 3(b)). Western blot indicated that IL-1β upregulated MMP-13 and downregulated collagen II and aggrecan in CHON-001 cells. However, these effects were reversed by ZEB1 silencing (Figure 3(c)). CCK-8 uncovered that ZEB1 knockdown rescued the suppressive impact of IL-1β on CHON-001 cell proliferation (Figure 3(d)). Additionally, TUNEL assay demonstrated that treatment of CHON-001 cells with IL-1β accelerated cell apoptosis, which was abolished by ZEB1 knockdown (Figure 3(e)). These findings revealed that silencing of ZEB1 accelerated cell viability and inhibited ECM degradation and apoptosis of IL-1β-stimulated chondrocytes.

miR-128-3p directly targeted ZEB1
Subsequently, using the StarBase database, ZEB1 was predicted as a target of miR-128-3p. The potential binding sequences between miR-128-3p and ZEB1 are shown in Figure 4(a). Then, a luciferase reporter assay was performed to verify that miR-128-3p mimics markedly suppressed the luciferase activity of ZEB1-WT. However, the luciferase activity was not affected in cells transfected with ZEB1-MUT (Figure 4(b)). The direct binding between miR-128-3p and ZEB1 was further confirmed by RIP assay. The results uncovered that the enrichment of ZEB1 and miR-128-3p was higher in the Ago2 group compared to IgG group (Figure 4(c)). miR-128-3p expression was decreased in CHON-001 cells transfected with miR-128-3p inhibitor (Figure 4(d)). Additionally, the mRNA and protein levels of ZEB1 was down-and upregulated in cells by miR-128-3p upregulation and downregulation, respectively (Figure 4(e,f)). ZEB1 was also significantly downregulated following miR-128-3p overexpression in IL-1β-activated CHON-001 cells (Figure 4(g)). Collectively, the data confirmed miR-128-3p could directly target ZEB1 and negatively regulate its expression.

Discussion
OA is a highly prevalent chronic joint disease [23]. This study confirmed that miR-128-3p level was reduced and that of ZEB1 was enhanced in OA.  Furthermore, miR-128-3p modulated cartilage ECM degradation, cell viability and apoptosis via ZEB1 in IL-1β-treated chondrocytes. Several miRNAs have been reported to be involved in the pathogenesis of OA [24,25]. miR-128-3p was found to be associated with inflammatory response and ECM degradation. For example, miR-128-3p could directly target SIRT1 to mediate the TNF-αinduced inflammatory response in bone marrow mesenchymal stem cells [26]. miR-128-3p also attenuated chondrocyte apoptosis, ECM degradation and inflammation via targeting Wnt-induced secreted protein 1 in OA [12]. Herein, we demonstrated that miR-128-3p was downregulated in chondrocytes and OA tissues. IL-1β is a cytokine that can induce a series of pathogenic responses in chondrocytes, thus playing a crucial role in the progression of OA [27]. Consistently, miR-128-3p expression was also suppressed in IL-1β-treated CHON-001 cells in the present study. Furthermore, miR-128-3p overexpression reduced the expression levels of MMP-13 and improved those of collagen II and aggrecan levels in IL-1β-activated CHON-001 cells. It was verified that MMP-13 was implicated in ECM degradation in OA. Cartilage ECM mainly consist of collagen II and aggrecan [28]. These findings indicated that upregulated miR-128-3p could attenuate cartilage ECM degradation in OA. Besides, miR-128-3p overexpression overturned the suppression on CHON-001 cell viability and the increased cell apoptosis caused by IL-1β.
ZEB1, a member of the ZEB family, is a known transcriptional regulator that plays critical roles in the induction of epithelial-to-mesenchymal transition and other biological processes [29,30]. It has been reported that ZEB1 is involved in the excessive production of ECM proteins via the transforming growth factor β1 signaling pathway in corneal endothelial cells [31]. Furthermore, NEAT1 promoted ECM progression via regulating the miR-27b-3p/ZEB1 axis in diabetic nephropathy [32]. Another study demonstrated that miR- 200b attenuated inflammatory responses via ZEB1 in gingival fibroblasts [33]. In the present study, ZEB1 was predicted as a downstream gene of miR-128-3p in IL-1β-induced chondrocytes. Silencing of ZEB1 suppressed ECM degradation and cell apoptosis and increased the viability of IL-1βtreated CHON-001 cells. In addition, ZEB1 was downregulated following miR-128-3p overexpression. Functional assays revealed that miR-128-3p mimics attenuated ECM degradation and cell apoptosis and increased the proliferation of IL-1βactivated CHON-001 cells, while ZEB1 overexpression restored these effects.

Conclusion
Our data demonstrated that miR-128-3p is overexpressed in patients with OA and can regulate OA cartilage ECM degradation, cell apoptosis and proliferation via targeting ZEB1 in chondrocytes. These findings indicated that miR-128-3p might be of value as a potential target for OA treatment.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.