miR-200a/b relieves IL-1β-induced cell injury in knee articular chondrocytes ex-vivo by targeting fucosyltransferase 4 (FUT4)

Background: Osteoarthritis (OA) is a common subtype of arthritis with prevalence increase with age, and is characterized by the degeneration of articular cartilage. Chondrocytes play curial role in the formation of the articular cartilage. This work aimed to gure out the effect of miR-200a/b in chondrocytes of OA, as well as the underlying molecular mechanism. Methods: Cell viability, apoptosis, pro-inammatory factors secretion, and matrix degradation were detected with cell counting kit-8 (CCK-8), ow cytometry, enzyme-linked immunosorbent assay (ELISA), and western blotting, separately. Expression of miR-200a/b and fucosyltransferase 4 (FUT4) was measured by RT-qPCR (RNA level) and western blot (protein level). The relationship between miR-200a/b and FUT4 was veried by dual-luciferase assay and RNA immunoprecipitation (RIP) assay. Results: Interleukin 1β (IL-1β) induced OA cell model in primary chondrocytes ex-vivo, as evidenced by cell viability inhibition, apoptosis rate promotion, and IL-6 and tumor necrosis factor α (TNF-α) resection enhancement, as well as Collegen 2a1 (Col2a1) and Aggrecan expression inhibition. Expression of miR-200a/b was downregulated in knee articular cartilage of OA patients and IL-1β-induced primary chondrocytes. miR-200a/b overexpression decreased IL-1β-induced cell injuries, which was further blocked by FUT4 upregulation. Mechanically, FUT4 was negatively regulated by miR-200a/b via target binding. Conclusion: miR-200a/b could alleviate IL-1β-induced chondrocyte injuries via targeting its downstream gene FUT4, suggesting that miR-200a/b-FUT4 axis might be a potential candidate to the treatment of OA.


Background
Osteoarthritis (OA) is classic age-related arthritis characterized by serious pain, limited movement, and crepitus [1]. The prevalence and severity of OA increase with age, thus OA has been becoming one common thereat in the old populations [2]. Synovium and articular cartilage degradation injury and a variable degree of in ammation are the mainly pathological changes of OA [3]. Articular chondrocyte is the only cell type in articular cartilage, therefore plays the pivotal role in the fate of articular cartilage [4].
The aberrant functions of chondrocytes result in the imbalance of extracellular matrix (ECM) synthesis [5], which lead to degradation of the articular cartilage tissue. Therefore, chondrocytes have been becoming as the preferred cells in OA researches, and apoptosis of chondrocytes is suggested to contribute to the pathogenesis of OA [6]. Although chondrocytes comprise 2-3% of total cartilage volume [4], it is necessary to study the cellular functions of chondrocytes for a better understanding of the pathogenesis of OA.
It has been widely suggested that the aetiology of OA was attributed to the deregulation of gene expression in chondrocytes [7]. microRNAs (miRNAs) belong to small non-coding RNAs that are essentially endogenous transcripts. Data indicated that miRNAs are direct regulators for about one third of the genes via target binding [8]. Recently, miRNAs has also been shown to be involved in maintaining cartilage homeostasis in OA, as well as in ammatory response during development [9]. For example, several miRNAs have been demonstrated to participate in OA, such as miRNA (miR)-320, miR-21 and miR-140 [10][11][12]. The miR-200 family is mainly characterized as tumor suppressor [13], and miR-200a-3p (miR-200a) and miR-200b-3p (miR-200b) have been preliminarily identi ed in OA [14,15]. However, the potential role of miR-200a/b in OA development remains to be widely studied.
The fucosyltransferase 4 (FUT4) is one member of FUTs family that are key enzymes in the surface of cells and take part in multiple biological processes, including in ammation and cancer progression [16,17]. In OA, FUT4 has been reported to be complicated in the progression of OA [18]. Interleukin 1beta (IL-1β) is a pro-in ammatory cytokine that contributes to the onset and progression of OA [19,20]. Therefore, we aimed to investigate the role of miR-200a/b in IL-1β-induced OA model in primary chondrocytes exvivo. The mechanism study was also performed to explore the targeted functional protein of miR-200a/b in modulating knee articular chondrocyte progression including cell progression, in ammation, and matrix degradation.

Patients and tissue specimens
With the written informed consents obtained from each patient, a total of 60 knee cartilage tissues were collected from 2016 to 2018 in the Baoji Traditional Chinese Medicine Hospital, including 30 OA specimens from OA patients (mean age: 42.5 years old) and 30 normal specimens from emergency traumatic amputated patients (mean age: 34.6 years old). All patients with arthritis, including rheumatoid arthritis (RA), OA and septic arthritis, were excluded in control group. The samples were put into liquid nitrogen at once and stored for cryopreservation. This study was approved by the ethic committee of the Baoji Traditional Chinese Medicine Hospital.

Cell culture
Under aseptic conditions, the cartilage tissues were removed of brous connective tissues, and cut into small pieces. After washing with sterile phosphate-buffered saline (PBS), cartilage tissues were digested with 0.25% trypsin (Invitrogen, Carlsbad, CA, USA) for 30 min, and then with 0.2% collagenase Type II (Col2; Millipore Corp., Billerica, MA, USA) in Dulbecco's modi ed Eagle's medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) for 10 h at 37 o C. Subsequently, these cells were ltered with 40 mm lter, and then centrifuged and washed at least three times, last re-suspended in growth culture media supplemented with DMEM (Gibco), 10% fetal bovine serum (Gibco) and 100 U/ml penicillin and 100 µg/ml streptomycin solution (Invitrogen). The cells were cultured in humidi ed sterile air with 5% (v/v) CO 2 at 37 o C, and the rst passage chondrocytes were obtained for 10 days. The chondrocyte cells were treated with 0.25% trypsin (Invitrogen) for cell passage.

Western blot
The total protein in IL-1β-treated chondrocytes was extracted by RIPA lysis buffer (Beyotime), and the concentration was determined using BCA™ Protein Assay Kit (Pierce, Rockford, IL, USA). 20 µg of proteins were separated on 8-12% SDS-PAGE and transferred onto PVDF membrane (Millipore). After blocking with 5% nonfat milk, membranes carrying proteins were incubated with primary antibodies and HRPconjugated secondary antibodies. β-actin was used as an internal standard to normalize protein level.

Statistical analysis
Data were presented as mean ± standard deviation (SD) from three independent experiments, and analyzed on Graphpad Prism 6.0 (GraphPad Software Inc., La Jolla, USA). Two tailed Student's t test was used to calculate statistical signi cance between two groups test, and one-way analysis of variance was for multiple groups. Result with P value < 0.05 was considered to be statistically signi cant.

Results
Expression of miR-200a/b was downregulated in OA cartilage tissues Then, RT-qPCR analysis was used to measure miR-200a/b expression in OA, and we found that relative miR-200a level was lower in this cohort of OA patients than control (Fig. 1A). Similar results were obtained about miR-200b expression status in the same OA patients (Fig. 1B). These data suggested a potential role of miR-200a/b in OA.

The cell injury induced by IL-1β in primary chondrocytes
Therefore, we established and veri ed the cell model of OA in chondrocytes. The primary chondrocytes were isolated and cultivated with 5, 10 and 20 ng/ml of IL-1β for 12 h ex-vivo. Then cell injury was measured. CCK-8 assay showed that 10 and 20 ng/ml of IL-1β exposure signi cantly induced a 38% and 57% cell viability inhibition, respectively ( Fig. 2A); ow cytometry determined that apoptosis rate was distinctively increased in response to IL-1β from 5.7-16.9% (10 ng/ml) and to 25.6% (20 ng/ml) ( Fig. 2B and 2C). Released pro-in ammatory cytokines IL-1β, IL-6 and TNF-α were dramatically upregulated when the primary chondrocytes were exposed with 10 and 20 ng/ml of IL-1β ( Fig. 2D and 2E). In addition, western blotting was utilized to assess cartilage matrix degradation, and the decrease of Col2a1 and Aggrecan indicated the degradation of chondrocyte matrix in response to 10-20 ng/ml of IL-1β (Fig. 2F). Then, expression of miR-200a/b in IL-1β-induced cell injury in this cell model of OA was clari ed. RT-qPCR analysis revealed that relative miR-200a and miR-200b levels were consistently downregulated in IL-1βtreated primary chondrocytes compared with control group (without IL-1β exposure) ( Fig. 2G and 2H). Taken together, IL-1β could induce in ammatory injury and matrix degradation in chondrocytes in a certain of dose-dependent manner.

FUT4 expression was directly modulated by miR-200a/b in chondrocytes
Recently, FUT4 had been reported to be aberrantly upregulated in OA cartilage tissues [18], we hypothesized FUT4 played an important role in knee articular chondrocytes. As predicted by DIANA Tools (http://DianaTools/microT_CDS), FUT4 3'UTR was predicted to be a potential target of both miR-200a and miR-200b ( Fig. 3A and 3B). Subsequently, this potential target binding relationship was further identi ed. With co-transfection, the relative luciferase activity of FUT4-WT was signi cantly reduced in 293T cells transfected miR-200a mimic or miR-200b mimic, compared with miR-NC mimic transfection ( Fig. 3C and 3D); whereas miR-200a/b mimic had no effect on the luciferase activity of FUT4-MUT. Moreover, the enrichment level of FUT4 was up to 4.6-fold in RIP-Ago2 derived from miR-200a mimictransfected primary chondrocytes (Fig. 3E); moreover, that was 6.4-fold enrichment of FUT4 in miR-200btransfected chondrocytes (Fig. 3F). These results indicated a target binding relationship between miR-200a/b and FUT4.
Next, the expression of FUT4 in IL-1β-induced knee OA model was gured out. Expression of FUT4 both on mRNA level and protein level was gradually upregulated by IL-1β in primary chondrocytes (  Fig. 5B and 5C). The highly expressed IL-1β, IL-6 and TNF-α in IL-1β-induced primary chondrocytes were attenuated by miR-200b restoration, and this effect was further blocked by FUT4 upregulation (Fig. 5D and 5E). On the contrary, the low expression of Col2a1 and Aggrecan was enhanced in chondrocytes under IL-1β stress when miR-200a was upregulated, and this promotion was counteracted by FUT4 (Fig. 5F). Notably, introduction of pcDNA-FUT4 vector could not only abolish the inhibitory effect of miR-200b upregulation on FUT4 expression (Fig. 6A), but also the suppressive role of miR-200b overexpression in IL-1β-induced cell viability inhibition, apoptosis, in ammation response, and matrix degradation (Fig. 6B-6F). Collectively, these data suggested that miR-200a/b could attenuate IL-1β-induced cell injury in primary chondrocytes, and this protective effect relied on directly inhibiting its downstream target gene FUT4.

Discussions
The therapies for OA patients were mainly to relieve the pain and in ammation [21]. Researches had shown that the important pathological feature of OA contained articular cartilage degeneration, which was largely attributed to apoptosis of chondrocytes [22]. At present, the medications were often accompanied with severe side effects. Therefore, studies of the basically molecular mechanism of chondrocytes on cytological level might contribute to a better understanding and novel therapy of OA [23]. In this study, we used the primary chondrocytes in knee articular and investigated the effect of miR-200a/b on IL-1β-induced chondrocyte injury. The results showed that IL-1β, as OA cell model stimulation, could evoke cell viability inhibition, apoptosis, in ammation and matrix degradation in primary chondrocytes ex-vivo, and miR-200a/b was downregulated in OA knee cartilage and IL-1β-induced chondrocytes. Functionally, restoration of miR-200a/b signi cantly alleviated IL-1β-induced knee articular chondrocyte cell injury through negatively regulating its downstream target gene FUT4.
It was generally believed that miR-200 family, consisting of ve miRNAs miR-141/200a/200b/200c/429 served pivotal role in cancer incidence [13]. Very recently, several members of miR-200 family had been declared to participate in OA progression. For example, miR-141 was associated with pathological destruction of cartilage through regulating cell migration and cell spreading of OA broblast-like synoviocytes, apoptosis of chondrocytes, and lipid metabolism [15,24,25]. Besides, serum miR-200c together with miR-100 and miR-1826 was proposed to be potential diagnostic biomarkers for knee OA [26]. Few studies had been published to clarify the potential function of miR-200a and miR-200b in OA pathogenesis. For instance, Wang et al. [27] discovered that forkhead box C1 positively modulate OA synovial broblast proliferation and pro-in ammatory cytokine production partially via regulated by miR-200a. In terms of miR-200b,Wu et al. [14] demonstrated that miR-200b was downregulated in OA chondrocytes and could serve as a repair factor for OA cartilage, because its upregulation signi cantly suppressed MMPs levels and promoted Col2 levels via declining DNMT3A expression. Luckily, we managed to verify the expression patter of miR-200a/b in knee cartilage of OA patients and IL-1β-induced OA chondrocytes model ex-vivo, which supported the ndings of previous researches [14,27]. What's more, FUT4 as a novel, common downstream target gene of miR-200a/b was came to light. Here, we performed functional experiments to detect the chondrocyte cell viability, apoptosis rate, production of pro-in ammation factors (IL-6 and TNF-α), and expression of ECM proteins (Col2a1 and Aggrecan).
FUTs were involved in signal transduction, in ammatory, tumor progression, and metastasis. For example, FUTs mediated the multidrug resistance of human hepatocellular carcinoma [16]. As for arthritis, accumulating evidences implied that FUTs had controlled the development of RA and juvenile idiopathic arthritis [28,29]. For example, FUT1 and FUT7 were upregulated in RA synovial broblast cell and synovial uid, respectively [28,29]. FUT1 and FUT2 mediated angiogenesis and cell adhesion in RA [29,30]. However, FUTs in OA had not been widely studied. Hu et al.
[18] revealed the expression pro le of FUTs, and mRNA expression of FUT1, FUT2, FUT3, and FUT4 were remarkably upregulated in healthy and OA human cartilage tissues, while FUT7 mRNA expression was downregulated. Furthermore, Li et al. [31] appointed that most of FUTs were higher in RA compared to OA synovial tissues, including FUT1, FUT2, FUT7 and FUT4. According to our ndings, FUT4 expression both on mRNA level and protein level were dramatically increased in IL-1β-induced OA cell model ex-vivo, and overexpression of FUT4 attenuated miR-200a/b-induced effects on the reduction of apoptosis and secretions of IL-6 and TNF-α. At the same time, more researches should be launched to explore which signaling pathway(s) underlies miR-200a/b/FUT4 axis-modulated chondrocytes progression, such as NF-κB [18] and MAPK/ERK [14] pathway.

Conclusion
In summary, our study demonstrates that miR-200a/b overexpression rescued cell viability, but alleviated apoptosis, in ammatory response and matrix degradation in IL-1β-treated primary chondrocytes; and high-expression of FUT4 blocked the protective effect of miR-200a/b in knee articular chondrocyte injury ex-vivo. This work suggested miR-200a/b-FUT4 axis as a potential candidate to improve chondrocyte viability in knee OA [32], even though further evidence should be obtained to verify the above conclusion in vivo and the underlying signaling pathway need more investigations to be fully disclosed.

Declarations
Acknowledgement None Funding

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Availability of data and materials The datasets analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contribution: All authors made substantial contribution to conception and design, acquisition of the data, or analysis and interpretation of the data; take part in drafting the article or revising it critically for important intellectual content; gave nal approval of the revision to be published; and agree to be accountable for all aspect of the work.

Ethics approval and consent to participate
The present study was approved by the ethical review committee of Baoji Traditional Chinese Medicine Hospital

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