MiRNA-6089 inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation and induces apoptosis by targeting CCR4

Abstract Several studies have suggested that fibroblast-like synoviocytes (FLSs) and miRNAs are implicated in the pathogenesis of rheumatoid arthritis (RA). This study was aimed to evaluate the function of miR-6089 in the regulation of RA-FLSs. The levels of miR-6089 were detected to be significantly lower in the synovial tissues and FLSs of RA than in the healthy synovial tissues and FLSs. The miR-6089 up-regulation in RA-FLSs significantly inhibited the proliferation and promoted cell apoptosis accompany with an increase protein expression of cleaved-Caspase-3, -8 and -9. Furthermore, CCR4 was determined to target miR-6089 directly, and its expression was significantly increased in the synovial tissues of RA than in the healthy synovial tissues. The overexpression of CCR4 reversed the effect of miR-6089 on proliferation and apoptosis in RA-FLSs effectively. In conclusion, our study suggests that the miR-6089 may be a potential target for prevention and treatment of RA.

progression in order to improve therapeutic strategies.
MicroRNAs (miRNAs) are endogenous small RNAs of about 20-24 nucleotides in length that can bind to the 3' untranslated region (UTR) of a target mRNA through complete or incomplete complementary base-pairing, and either degrade the transcripts or inhibit their translation [7]. Although miRNAs comprise only 2% of the human genome, they regulate the expression of nearly one-third of the genes, especially those involved in cell differentiation, cell proliferation and apoptosis and immune response [8]. Recently, a variety of abnormally expressed miRNAs have been found in RA peripheral blood mononuclear cells and FLS [9], but the specific mechanism involved in regulating this process and the mechanism by which abnormally expressed miRNAs are involved in regulating the occurrence of RA development is unclear.
Therefore, in this study, we detected the expression of miR-6089 in synovial tissues of healthy and RA and FLSs, and evaluated the effects of miR-6089 on proliferation, apoptosis and inflammatory response in RA-FLS. In addition, we also explored the role of miR-6089 in RA-FLSs, and the underlying molecular mechanisms were determined in order to provide a basis for future clinical applications for RA.

Materials And Methods Samples of tissues
The synovial tissues from 25 RA patients were obtained during knee replacement surgery. RA was diagnosed according to the American College of Rheumatology criteria for classification of disease.
Healthy synovial tissues from 16 patients with a traumatic knee condition were collected as the normal control. The study was approved by the Ethics Committee of The Third Clinical College of Wenzhou Medical University, Wenzhou People's Hospital, and all subjects provided informed consent.

Cell Culture And Transfection
The normal human FLSs and RA-FLSs were obtained from Cell Applications (San Diego, CA, U.S.A.).
Following overnight incubation, 10 µL CCK-8 reagent was added to each well and the absorbance at 450 nm was determined after culturing for 24 h, 48 h and 72 h according to the manufacturer's protocol.

Cell Apoptosis
The cells were collected, washed twice with cold 1 × PBS and resuspended in 100µL binding buffer, followed by incubation with Annexin V-FITC and propidium iodide for 15 min at room temperature in the dark. Next, 200µL of binding buffer was added, and the cells were analyzed by flow cytometry.

Luciferase Reporter Assay
According to TargetScan (http://www.targetscan.org) and MiRanda (http://www.microrna.org/microrna/home.do) databases, CCR4 was predicted as a putative target of miR-6089, which was verified using the dual luciferase reporter assay. Briefly, 2 × 10 4 cells/96 well in the logarithmic growth phase was cultured overnight, then 100 ng WT-CCR4-3'UTR, and MUT-CCR4-3'UTR luciferase reporter plasmid was co-transfected with 50 ng miR-6089 mimic or NC using Lipofectamine 3000, and the cells were cultured for 48 h. The relative luciferase activity was detected according to the dual luciferase assay kit instructions. Five replicates were tested per condition.

qRT-PCR analysis
Total RNA was extracted from collected cultured FLSs and tissue samples utilizing a Trizol reagent (Invitrogen, Carlsbad, CA), and the purity and concentration of RNA were determined with a spectrophotometer. The RNA was reversely transcribed into cDNA templates used for real-time RT-PCR, which was then performed on an 7500 real-time PCR system (ABI, Foster City, CA) utilizing a SYBR Premix kit (TaKaRa, Tokyo, Japan) to measure the relative expression of, miR-6089, and CCR4

Elisa Assays
The concentrations of MMP-1, TNF-α and IL-6 in the cell culture supernatant were detected using ELISA kits (Affymetrix, Santa Clara, CA, USA) following the manufacturer's recommendations.

Western Blot Analysis
To prepare protein extracts, harvested cells were washed thrice with chilled PBS, and lysed with RIPA buffer supplemented with protease inhibitors. The extracted protein was quantified using the BCA method, and the cell lysates were diluted in 5 × loading buffer at the ratio of 1:4. After denaturing at 95 °C for 10 min, 30 µg protein per sample was subjected to SDS-PAGE. The protein bands were transferred to PVDF membranes and the latter were blocked with 5% skim milk at room temperature for 2 h. The blots were incubated overnight with primary antibodies against CCR4 (1:1000), Caspase-3(1:1000), Caspase-8(1:1000), Caspase-9 (1:1000), and GAPDH (1:5000) at 4 °C on a shaker. After washing thrice with TBST buffer, the blots were probed with HRP-conjugated secondary antibody (1:5000) for 1 h at room temperature, followed by 3 washes with TBST. ECL chromogenic substrate was used to visualize the bands, and the Image J software was used to determine the gray value of each band, and the content of each protein was calculated relative to that of GAPDH.
Statistical Analyses SPSS 19.0 was used for statistical analysis. Descriptive statistics were used to determine the group means and standard deviations in the numerical data. Independent sample t-test was used to compare two groups, and one-way ANOVA along with Dunnett's or Bonferroni's test was used for multiple groups. The level of significance was set to p < 0.05.

miR-6089 was down-regulated in RA synovial tissues and FLSs
The expression levels of miR-6089 were significantly lower in synovial tissues of RA patients versus the healthy controls (P < 0.01; Fig. 1A). Consistent with the patient samples, the human RA-FLSs showed dramatically lower levels of miR-6089 compared to the normal human FLSs (P < 0.01; Up-regulation of miR-6089 suppresses the proliferation and promotes the apoptosis in RA-FLSs To determine the effects of miR-6089 on RA-FLSs proliferation, and apoptosis, miR-6089 mimic was firstly used to transfect RA-FLSs with the miR-6089 mimic, which showed a 5.7-fold increase in miR-6089 expression (P < 0.01; Fig. 2A). The results from CCK-8 assay suggested that miR-6089 overexpression markedly suppressed the proliferation of RA-FLSs (Fig. 2B).
Next, flow cytometry was performed to detect the apoptosis after miR-6089 overexpression in RA-FLSs, indicating that miR-6089 overexpression significantly increased apoptosis ratio compared to the miR-NC group (P < 0.01; Fig. 2C). Mechanistically, we also found the increased protein expression of caspase-3, -8 and − 9 in miR-6089 overexpressing RA-FLSs (Fig. 2D). Overall, our findings indicated that miR-6089 could inhibit the proliferation and trigger apoptosis of RA-FLSs.
Up-regulation of miR-6089 inhibits the release of inflammatory cytokines in RA-FLSs Next, we determined the role of miR-6089 in the response to the inflammatory cytokines of RA-FLSs.
As shown in Fig. 3, miR-6089 overexpression significantly the release of the MMP-1, TNF-α and IL-6, suggesting that miR-6089 might protect the RA though inhibiting the inflammatory secretion.

Mir-6089 Acts Directly On Ccr4 In Ra-flss
The TargetScan database predicted that the 3'-UTR of the CCR4 gene was complementary to miR-6089 indicating that CCR4 is a putative target of miR-6089 (Fig. 4A). As shown in Fig. 4B, the possible relationship was validated by dual luciferase gene reporter assay which showed that the luciferase activity markedly reduced when co-transfected with the miR-6089 mimic and WT CCR4, whereas cotransfection with miR-6089 mimic and MUT CCR4 had no significant effect on the luciferase activity.
Moreover, In contrast with the miR-NC group, there was a lower level ofCCR4 in miR-6089 overexpression group on both the mRNA and protein levels (Fig. 4C, D). Interestingly, CCR4 expression was significantly increased in RA synovial tissues (Fig. 4E). These results suggested that CCR4 is a target gene of miR-6089 and is negatively regulated by the latter.
Knocking down of CCR4 inhibits the proliferation and promotes the apoptosis in RA-FLSs First, the CCR4 expression in RA-FLSs was silenced by transfection of siRNA of CCR4. Two independent CCR4 siRNAs were used in case of off-target effect. Both siRNAs successfully knocked down CCR4 expression, with a scrambled sequence as control (Fig. 5A). The proliferation was detected with the CCK8 assay, which demonstrated that silencing CCR4 could significantly inhibit the cell proliferation (Fig. 5B). Flow cytometry was carried out 48 hours after the transfection of siRNAs.
Both siRNAs of CCR4 increased the cell apoptosis (Fig. 5C) Dysregulation of miRNAs is established to be involved in the many diseases. miR-6089 serves a tumorsuppressive miRNA in ovarian cancer, which directly targets MYH9 to inactivate the Wnt/βcatenin and its downstream EMT [17]. In our study, we proved that miR-6089 was significantly decreased in synovial tissues and FLS of RA patients compared with health donor. In vitro studies further showed that the recovery of miR-6089 significantly inhibited the proliferation accompany with an increased apoptosis in RA-FLSs. These results indicated that miR-6089 might play an important role in progression of RA. Further, the overexpression of miR-6089 notably increased the apoptosis with the apoptosis-related protein expression of caspase-3, -8 and − 9 in RA-FLSs. Moreover, we found a lower level of inflammatory cytokines in RA-FLSs transfected with miR-6089 mimic compared with the negative control. Thus, miR-6089 processes the inhibitive effect in the development of RA-FLSs.
Although the etiology and pathological mechanism of RA is still unknown, more and more reports confirm that miRNAs are closely related to the pathological process of RA [18,19]. Different miRNAs can target and regulate different target genes to affect the pathological process of RA [20]. Thus, to further understand the underlying mechanisms by which miR-6089 inhibits the growth of RA-FLSs, we sought to identify its direct target genes. Bioinformatics analysis predicted CCR4 as a target of miR-62089, and the dual luciferase gene reporter assay further confirmed that CCR4 was negatively regulated by miR-6089. Meanwhile, in RA-FLSs, miR-6089 overexpression markedly decreased CCR4 expression. All of these results indicated the targeted binding correlation between miR-6089 and CCR4 in RA-FLSs.
Recently, studies showed that CCR4 is highly expressed on circulating Tregs [21]. Tregs is recruited at tumor sites in many cancers, such as breast cancer [22] and colorectal cancer [23]. The anti-CCR4 antibody, alone or in combination with other immune modulators, may potentially be used in the treatment of a human solid cancer with high levels of CCR4 expression in tumor-infiltrating leukocytes and abnormal plasma CCR4 ligand levels [24]. Importantly, CCR4 has been widely implicated in the pathogenesis of inflammatory diseases such as asthma and atopic dermatitis due to its expression on Th2 cells [25].Interestingly, it was found an increased mRNA level of CCR4 in synovial tissues. Notably, overexpression of CCR4 reversed the effects of miR-6089 on proliferation and apoptosis in RA-FLSs by. Our results showed that miR-6089 performed its inhibitive role in RA-FLSs, at least in part, by targeting CCR4.
In conclusion, miR-6089 is found to be frequently downregulated in RA synovial tissues and FLSs. And the overexpression of miR-6089 suppresses proliferation, promotes apoptosis associated with the apoptosis protein expression (caspase-3 -8 and − 9), as well as decreases the secretion of inflammatory cytokines in RA-FLSs by targeting CCR4. Our findings provide new insights into the roles of miR-6089 and CCR4 in RA, and identify potential prognostic biomarkers and therapeutic targets.

Funding
This study was supported by Wenzhou Science and Technology Bureau Project (Y20160301 and 2017Y0539).

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.