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MiR-498 suppresses proliferation and inflammation in fibroblast-like synoviocytes in rheumatoid arthritis via targeting JAK1

Purpose: To investigate the effect of microRNA 498 (miR-498) on proliferation and inflammation of rheumatoid arthritis (RA) fibroblast-like synoviocytes (RA-FLSs) in rheumatoid arthritis (RA). Methods: MiR-498 level was evaluated in both RA synovial tissues and RA-FLSs using real-time polymerase chain reaction (PCR). MicroRNA-498 overexpression or knockdown was performed in RAFLSs. Proliferation, apoptosis, cell cycle and inflammation induced by miR-498 mimics or inhibitor were used to explore the function of miR-498 in RA. Results: Expression level of miR-498 was downregulated in both RA synovial tissues and RAFLSs. MicroRNA-498 mimics decreased proliferation and arrested cell cycle, whereas miR-498 inhibitor caused the opposite effects in RA-FLSs. In addition, miR-498 mimics suppressed inflammation and promoted cell apoptosis, while miR-498 inhibitor promoted inflammation and inhibited cell apoptosis in RA-FLSs. Furthermore, the effect of miR-498 on the proliferation, inflammation and apoptosis of RAFLSs was mediated by its ability to target and downregulate JAK1. Conclusion: These results indicate that miR-498 inhibits the proliferation and inflammatory responses of RA-FLSs by targeting JAK1, thus revealing a new therapeutic target for RA treatment.


INTRODUCTION
Rheumatoid arthritis (RA), a chronic autoimmune disease, is characterized by inflammation and destruction of the joints [1,2]. It reduces the quality of life, produces loss of functionality, and increases morbidity and mortality [2]. The incidence of RA has reached 0.5-1 % worldwide [3]. The main clinical manifestations involved in the symmetrical joint are arthralgia, redness, swelling, and limited range of motion [4]. Current RA treatment is focused on blocking inflammation early in the disease course [2]. In addition, the main goals of RA treatment are to reduce joint and organ damage, improve physical function, and prevent long-term complications [2]. Fibroblast-like synoviocytes (FLSs) in the synovial intimal lining play a crucial role in RA pathogenesis [5]. Therefore, exploring the mechanism underlying RA treatment in RA-FLSs is of great importance. MicroRNAs (miRNAs), small non-coding RNAs, are single-stranded RNAs of 19-25 nucleotides in length which cause mRNA degradation and protein activity reduction by binding to the 3ʹ-untranslated region (UTR) of the target mRNA [6][7][8][9]. MicroRNAs have been reported to be associated with RA-FLSs. For example, miR-708-5p is downregulated in the synovial tissues of patients with RA. On the other hand, miR-708-5p induces cell apoptosis and suppresses colony formation and migration in RA-FLSs [10]. Moreover, the expression of miR-199a-3p is low in RA-FLSs, which results in decreased proliferation and elevated apoptosis [11]. Interestingly, miR-498 is associated with RA susceptibility [12], suggesting that miR-498 may be involved in RA-FLS progression. Janus kinase 1 (JAK1) is associated with the RA progression due to its role in cytokine signaling [13,14], and it regulates RA-FLSs. Previous studies have found that apoptosis of RA-FLSs can be induced via the JAK1-mediated signaling cascade [15]. In addition, JAK1 is involved in the activation of RA-FLSs in response to the inflammatory cytokines interleukin 6 and oncostatin M [16]. Although JAK1 is reported to have a role in RA, the functions of miR-498 and JAK1 in RA-FLSs have not been investigated. This study first demonstrated the effect of miR-498 on the proliferation and inflammation of RA-FLSs via targeting JAK1. Furthermore, this work will be useful to explore new therapeutic targets of RA.

EXPERIMENTAL Human samples
Thirty-two RA synovial and normal tissues from Zhejiang Hospital were collected, and they were divided into two groups: a normal group (n = 10) and an RA group (n = 22). All patients provided informed consent. The research protocols were approved by the Clinical Research Ethics Committee of Zhejiang Hospital (Approval no.2017-c-076) and World Medical Association Declaration of Helsinki.

Cell culture and transfection
The RA-FLSs were isolated from RA synovial tissues and were grown in RPMI 1640 medium containing 10% fetal bovine serum (FBS), and 293T cells were grown in Dulbecco's modified eagle medium supplemented with 10 % FBS. On the other hand, miR-498 mimics (miR-498), scrambled mimic control (Control), miR-498 inhibitor (miR-498-inh), and inhibitor negative control (NC-inh) were purchased from RiboBio (Guangzhou, China). The RA-FLSs were then transfected with these RNAs using Lipofectamine 2000 (Life Technologies Corp., Carlsbad, CA, USA). All cells were cultured in a humidified incubator with 5% CO 2 at 37°C.

Cell proliferation assay
Cell proliferation was assessed using MTT assay kit. Cells of 2 x 10 4 RA-FLSs were first seeded into 96-well plates. Thereafter, 10 μL of MTT solution was added (Dojindo Molecular Technologies, Gaithersburg, MD) per well at different periods of time, followed by incubation for another hour at 37°C. The absorbance was measured at each time point using a microplate reader.

Western blotting
Proteins from RA-FLSs were extracted and then quantified using BCA Protein Assay Kit (Pierce, Appleton, WI, USA). Equal amounts of proteins were separated using sodium dodecyl sulfatepolyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked using blocking buffer and incubated with specific primary antibodies overnight at 4 °C. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 37 °C, followed by visualization of the bands. The corresponding primary antibodies used were: Cyclin D1 (1:1000; Santa Cruz Biotechnology, CA, USA), p27

Cell cycle analysis
The transfected cells were collected and washed thrice with cold phosphate-buffered saline (PBS). Next, they were re-suspended in pre-cooled 75% ethanol and incubated overnight at 4 °C for fixation. The ethanol was then discarded, and 500 µl of PBS containing 20 µl of RNase A (100 µg/mL) was added. After incubation for 30 min, 400 µl of propidium iodide (PI; 50 µg/mL) was added to the cells drop-wise, followed by incubation for another 30 min. The cell cycle distribution was evaluated using FACScan flow cytometry (BD Biosciences, San Jose, CA, USA).

Cell apoptosis assay
Cell apoptosis was measured using the Annexin V-FITC/PI apoptosis detection kit (BD Pharmingen, San Jose, CA, USA). The cells were collected after transfection with miR-498 mimics or miR-498 inhibitor. They were then washed and re-suspended with 100 μL of binding buffer.
Next, Annexin V-fluorescein isothiocyanate (FITC) and PI were added. After incubation for 15 min, the stained cells were detected using FACScan flow cytometry (BD Biosciences, San Jose, CA, USA).

Statistical analysis
Data are reported as mean ± standard deviation (SD). Student's t-test was used to analyze differences between two groups. One-way analysis of variance (ANOVA) followed by Tukey's test was applied for multiple comparisons, and p < 0.05 was considered statistically significant.

Expression of miR-498 in RA synovial tissues and RA-FLSs
To examine miR-498 expression, the RA samples were harvested. As shown in Table 1, patients with RA were characterized based on parameters such as age, sex, duration of symptoms, rheumatoid factor (RF) positivity, anticyclic citrullinated peptide (anti-CCP) titer, and disease activity score 28 (DAS28). Next, miR-498 expression was evaluated using real-time PCR. The results showed that miR-498 was significantly down-regulated in RA synovial tissues when compared with that in normal tissues (Figure 1 A, p < 0.05). Similarly, miR-498 was decreased in RA-FLSs compared with that in FLSs (Figure 1 B, p < 0.05). Thus, the expression of miR-498 is inhibited in RA synovial tissues and RA-FLSs.

MiR-498 inhibits proliferation in RA-FLSs
To further investigate the function of miR-498 in RA-FLS proliferation, this study carried out miR-498 overexpression and knockdown experiments. First, real-time PCR indicated that miR-498 mimics and inhibitor were successfully expressed in RA-FLSs. The relative levels of miR-498 due to transfection with mimics were significantly increased compared with that of the Control (Figure 2A, p < 0.05). Conversely, the introduction of miR-498 inhibitor in the RA-FLSs significantly reduced the expression of miR-498 when compared with the introduction of NC-inh (Figure 2 A, p < 0.05). Next, the MTT assay revealed that miR-498 mimics decreased proliferation, whereas miR-498 inhibitor increased proliferation in RA-FLSs (Figure 2 B). Western blot assay further demonstrated that miR-498 mimics decreased the level of Cyclin D1 and increased the level of p27 in RA-FLSs. However, miR-498 inhibitor induced the reverse effect on the Cyclin D1 and p27 protein levels (Figure 2 C). Furthermore, to study the cell cycle distribution of RA-FLSs, cell cycle profiles of miR-498 mimics or inhibitor cells were measured using flow cytometry. As shown in Figure 2

JAK1 is a target of miR-498
To further explore the molecular mechanism of miR-498 in RA, it was predicted that miR-498 binds to JAK1 (Figure 4 A). Dual luciferase assay confirmed that miR-498 mimics significantly reduced the relative luciferase activity in JAK1 3'UTR-wt, whereas no significant change was found in JAK1 3'UTR-mut (Figure 4 B). Moreover, western blotting revealed that miR-498 mimics suppressed JAK1 protein levels while miR-498 inhibitor enhanced JAK1 levels (Figure 4 C). Together, these data showed that JAK1 is the target of miR-498 and its levels could be affected by miR-498.

MiR-498 regulate proliferation, inflammation, and apoptosis by inhibiting JAK1 in RA-FLSs
This study further investigated how miR-498 affected proliferation, inflammation, and apoptosis. MTT assay showed that miR-498 mimics inhibited cell proliferation in RA-FLSs. However, the effect was alleviated in RA-FLSs co-transfected with miR-498 mimics and JAK1 (Figure 5 A). ELISA indicated that miR-498 mimics repressed TNF-α and IL-1β levels, whereas miR-498 mimics and JAK1 overexpression reversed these levels (Figure 5  B). Additionally, western blotting determined that the decrease in the JAK1 and Cyclin D1 levels and the increase in p27 protein levels induced by miR-498 mimics could be reversed in the presence of JAK1. Similarly, miR-498 mimics and JAK1 overexpression reversed the reduction in Bcl-2, and the elevation of Bax and cleaved caspase-3 protein levels (

DISCUSSION
In this study, miR-498 expression was first evaluated in RA synovial tissues and RA-FLSs.
To investigate the function of miR-498 in RA, this study carried out miR-498 overexpression or knockdown experiments in RA-FLSs. = microRNA-498 was found to inhibited proliferation, cell cycle, and inflammation, and enhanced cell apoptosis in RA-FLSs. Janus Kinase 1 was predicted to be a target of miR-498. The interaction between miR-498 and JAK1 was also verified. The effect of miR-498 mimics on proliferation, cell cycle, inflammation, and apoptosis was alleviated in RA-FLSs cotransfected with miR-498 mimics and JAK1. These findings indicate that miR-498 may regulate the proliferation and inflammation of RA via targeting JAK1. Previous evidence has demonstrated that miR-498 is down-regulated in CD4+ T cells from the synovial fluid or the peripheral blood of patients with RA [17]. This study found a low miR-498 expression level in RA synovial tissues and RA-FLSs. In addition, Xiang et al. showed that miR-498 was associated with peripheral blood mononuclear cells from RA patients and could lead to the inhibition of cell differentiation in RA patients [18]. These data suggest that miR-498 may show a suppressive effect in RA-FLSs. Moreover, miR-498 mimics was demonstrated to inhibit proliferation, whereas miR-498 inhibitor elevated proliferation in RA-FLSs.
Moreover, miR-498 mimics arrested the cell cycle and induced apoptosis in RA-FLSs, as evidenced by the accumulation of G1 cells, inhibition of Cyclin D1 and Bcl-2 expression, and enhancement of p27, Bax, and cleaved caspase-3 protein expression. In addition, in allergic rhinitis patients, the level of miR-498 was enhanced and was associated with the allergic inflammation of nasal mucosa [19]. MicroRNA-498 mimics could inhibit inflammation, accompanied by the reduced production of TNFα and IL-1β. In contrast, miR-498 inhibitor promoted inflammation, leading to the elevation of TNF-α and IL-1β levels. Taken together, these results suggest that miR-498 may play an important role in the regulation of cell proliferation and inflammation in RA.
Accumulating evidence has shown that baricitinib, an oral selective inhibitor of JAK1, could be useful for RA treatment [20]. Furthermore, previous studies have found that suppressed of JAK1 and JAK3 phosphorylation and subsequent expressions of Stat1 and Stat1inducible genes, thus contributing to efficient RA therapy [21]. Tofacitinib was also reported to block the IL-6 receptor, leading to JAK/STAT pathway alteration in RA [22]. On the other hand, upadacitinib, a selective inhibitor of JAK1, played an important role in the signal transduction of IL-6, as well as in interferon-α/β and interferon-γ signal transduction [23]. Interestingly, in this study, JAK1 was inhibited in miR-498 mimic RA-FLSs. JAK1 overexpression in miR-498-mimic expressing RA-FLSs reversed the decrease in proliferation as well as TNF-α and IL-1β secretion induced by miR-498 mimics. In addition, miR-498 mimics and JAK1 overexpression promoted the cell cycle and suppressed apoptosis, resulting in the reduction of cells in the G1 stage, increase in cyclin D1 and Bcl-2 levels, and decreases in p27, Bax, and cleaved caspase-3 protein levels in RA-FLSs.
Notably, JAK1 was found to be a target of miR-498 in this study. These data suggest that miR-498 may function as a mediator of cell proliferation and inflammation via targeting JAK1 in RA. Taken together, miR-498 is expressed in low levels in RA tissues and RA-FLSs. miR-498 could affect cell proliferation and inflammation by targeting JAK1 in RA. This study provides new protential strategy in RA treatment. However, miR-498 may bind more target genes, which may be involved in cell proliferation and inflammation in RA. Moreover, the effect of miR-498 on the JAK-STAT signaling pathway in RA has not yet been investigated. Hence, in the future, further study will be conducted to investigate the mechanism of miR-498 regulation of cell proliferation and inflammation in RA through the JAK-STAT signaling pathway.

CONCLUSION
The findings of this work indicate that miR-498 suppressed the proliferation and inflammatory activity of RA-FLSs by targeting JAK1, thus identifying miR-498 as a promising therapeutic target for RA treatment.

Conflict of interest
The authors declare that no conflict of interest is associated with this work. No funding was received for the work.

Contribution of authors
We declare that this work was done by the researchers listed in this article. All liabilities related with the content of this article will be borne by the authors. LC and XZ designed all the experiments and revised the paper. HM and FY performed the experiments. LC and XZ wrote the paper.

Open Access
This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/rea d), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.