MicroRNA-15a/16/SOX5 axis promotes migration, invasion and inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes

Reciprocal repression between miR-15a/16 and SOX5

Using Target Scan 7.2 (http://www.targetscan.org/), we predicted that the putative miR-15a/16 target site should be located within the position 259-265 of SOX5 3’UTR. This binding sites in SOX5 3’UTR were conserved among species (Figure 1A). To test whether SOX5 is a functional target of miR-15a/16, the SOX5 3’UTR containing wild type (WT) and mutant (MUT) miR-15a/16 binding sites were subcloned into the luciferase reporter vector. Co-transfection with the WT SOX5 3’UTR and miR-15a/16 mimic into 293T cell for 48hrs significantly decreased the luciferase intensity of SOX5 3’UTR (Figure 1B). In contrast, co-transfecting with the mutant SOX5 3’UTR and miR-15a/16 mimic displayed no effect on luciferase activity (Figure 1B).

We then assessed the capability of miR-15a/16 to negatively regulate SOX5 expression. RA-FLSs cell line of MH7A was transfected with miR-15a/16 mimics for 48hrs. Endogenous SOX5 expression were then detected by RT-qPCR and western-blot. As shown in Figure 2, the mRNA (Figure 2A) and protein levels (Figure 2B, 2C) of SOX5 markedly decreased, followed by miR-15a or 16 overexpression compared with the miR-control transfection. Taken together, our data suggest that miR-15a/16 could directly bind to SOX5 3’UTR and inhibit SOX5 expression, implying a posttranscriptional mechanism of regulating SOX5 in RA-FLSs.

To determine whether miR-15a/16 is regulated by SOX5, we detected miR-15a/16 expression after SOX5 overexpressed in FLSs by transfecting with Ad-SOX5 into MH7A. SOX5 overexpression significantly decreased miR-15a (Figure 2D, p = 0.02) and 16 (Figure 2E, p = 0.01) expression levels in MH7A compared with the control groups. These data suggested that there might be a reciprocal repression between SOX5 and miR-15a/16.

Expression of miR-15a/16/SOX5 axis in primary FLSs from RA patients

We previously reported higher SOX5 expression in synovial tissue of RA patients than those in OA patients [4]. To further ascertain the relationship between miR-15a/16 and SOX5 expression in RA patients, we therefore used RT-qPCR to simultaneously examine miR-15a/16 and SOX5 expression in primary FLSs of RA and OA patients. The expression of miR-15a and 16 significantly decreased (p = 0.005 and 0.002, respectively, Figure 3A), while SOX5 levels (p = 0.001, Figure 3B) markedly increased in RA-FLSs (n = 10), as compared with those of OA patients (n = 10). A strong negative correlation existed between the expression levels of miR-15a and SOX5 in RA-FLSs (p = 0.01, r² = 0.62, Figure 3C). A similar negative correlation trend was also found between miR-16 and SOX5, but did not reach the statistical difference (p = 0.06, r² = 0.36, Figure 3C). These data suggest the down-regulated miR-15a might link to an increase in its target gene SOX5 expression in RA-FLSs, supporting that the negative feedback loop between miR-15a/16 and SOX5 axis participates in RA pathogenesis.

Effect of miR-15a/16/SOX5 axis on RA-FLSs migration and invasion

In our previous data, SOX5 showed the potent ability to prompt migration and invasion of RA-FLSs [4]. Thus, we sought to determine whether lower expression of miR-15a/16 contributes to the pathologic behavior in RA-FLSs, and whether it does so via SOX5. MH7A were transfected with miR-15a or miR-16 mimics for 48hrs. As shown in Figure 4, overexpression of miR-15a and 16 in RA-FLSs decreased cell migration by 60% (p = 0.0001) and 34% (p = 0.002) respectively compared with the controls (Figure 4A above and Figure 4C). Similarly, an in vitro invasion assay showed that miR-15a and 16 mimics significantly inhibited invasiveness of RA-FLSs by 62% and 43%, respectively (Figure 4A below and Figure 4D). Consistent results of miR-15a/16 overexpression suppressing cell migration and invasion were validated in primary RA-FLSs (n=3) (Supplementary Figure 1).

Cell migration and invasion are commonly accompanied by dynamic reorganization of the actin cytoskeleton. We examined the rearrangements of actin cytoskeleton in RA-FLSs after transfection with miR-15a/16 using F-actin staining. Compared with the controls (Figure 4B), miR-15a mimics markedly decreased stress fibers’ appearance and lamellipodia formation in RA-FLSs. However, miR-16 overexpression did not markedly affect the cytoskeletal reorganization in RA-FLSs (Figure 4B). Cell proliferation was also measured by CCK-8 assay. miR-15a and miR-16 overexpression had no effect on cell proliferation at each time point from 0 to 72hrs (data not shown).

The data above suggest that overexpression miR-15a/16 resulted in an inhibitory role on FLSs. We then performed rescue experiments. Following transfecting with Ad-SOX5 into MH7A for 72hrs, low migration (Figure 4E) and invasion of FLSs (Figure 4F) by miR-15a/16 mimics transfection was markedly alleviated by SOX5 overexpression. Taken together, these data suggest that miR-15a/16/SOX5 axis participated in driving RA-FLSs migration and invasion.

Regulation of miR-15a/16/SOX5 axis on cytokine production in RA-FLSs

Since SOX5 was involved in regulating IL-6, MMP-3, and MMP-9 expression in RA-FLSs in our previous studies [4], we subsequently tested the effect of miR-15a/16/SOX5 axis on inflammatory response. In addition to IL-6, MMP-3, and MMP-9, a series of cytokines, including IL-1β, IL-17, TNF-α, and MMP-1, which were highly expressed in RA synovial tissues and contributed to the invasive ability of RA-FLSs [2]. Expression of these cytokine were detected by RT-qPCR in RA-FLSs. Overexpression of miR-15a markedly inhibited IL-1β production (p = 0.04) in FLSs but did not affect any other cytokines expressions (Figure 5A, 5B). miR-16 mimics showed the ability to significantly suppress IL-1β (p = 0.05) and TFN-α(p = 0.02) expression in RA-FLSs compared with miR-control transfection group (Figure 5A, 5B). These data indicated that miR-15a/16 could decrease cytokines production in RA-FLSs. Furthermore, miR-15a/16 mimics-medicated lower IL-1β or TFN-α expression could also be blocked by SOX5 overexpression (Figure 5C, 5D). In primary RA-FLSs, SOX5 also showed the ability to rescue the lower IL-1β and TFN-α expression was caused by miR-15a/16 mimics transfection (Supplementary Figure 2). These results suggested a crucial role of miR-15a/16/SOX5 in the inflammatory process in RA-FLSs.

Association miR-15a/16 with treatment response in RA patients

We previously failed to identify the significant correlation between serum SOX5 levels and RA disease activity score of DAS28. We then addressed whether miR-15a/16 could function as a biomarker for predicting RA disease activity or therapy response. We first examined the expression miR-15a/16 in synovial tissues from responders and non-responders of DMARDs therapy (non-responders are defined as DAS28>3.2 after more than three months of DMARDs treatment). Expressions of miR-15a and 16 decreased more significantly in non-responders than those in responders (p = 0.03 and 0.04, respectively, Supplementary Figure 3), implying that miR-15a and 16 levels in synovial tissues might be related to therapy response.

An ideal biomarker should be obtained easily. We therefore tested the circulating miR-15a/16 levels in serum from DMARDs naïve RA patients and HC. The baseline serum miR-15a/16 expression levels in RA showed neither the statistical difference with those in HC serum nor the strong association with RA disease activity score of DAS28 (data not shown). However, we found that miR-15a/16 levels significantly increased after 3 months of DMARDs therapy (Figure 6A, 6B) as compared with baseline, following up the average DAS28 decreased from 5.1 at baseline to 3.6 at 3 months. We divided RA patients into responders and non-responders based on whether their DAS28 score has changed ≥1.2 after 3 months of DMARDs therapy. We demonstrated that the baseline circulating miR-15a levels was higher in responders than those in non-responders (p = 0.04) (Figure 6C). The baseline miR-16 levels also increased in responders as compared with non-responders, but did not reach statistical significance (Figure 6D). Accompanied by the decreased disease activity at 3 months after DMARDs treatment, serum miR-15a (Figure 6E, left) and miR-16 (Figure 6F, left) levels increased significantly from baseline to 3 months (p = 0.0006 and 0.04, respectively) in responders; however, the non-responders retained similar low miR-15a/16 levels at 3 months as compared to baseline (p = 0.34 and 0.84, respectively) (Figure 6E, 6F, right). These data suggest that low serum levels of miR-15a/16 at baseline are associated with inadequate response of 3 months DMARDs treatment in RA patients.

Patients

A total of 32 newly diagnosed RA patients were included in this study. RA patients and healthy controls (HC, n = 20) were recruited from the First Affiliated Hospital of Nanjing Medical University. The criteria for patient eligibility were: (i) fulfillment of the revised 1987 American Rheumatism Association criteria for RA; (ii) naïve to DMARDs or biologics treatment; (iii) with active disease (defined as Disease Activity Score of 28 joints [DAS-28] > 3.2). This study was approved by the First Affiliated Hospital of Nanjing Medical University Committee on Human Research. All subjects in this study provided written informed consent. Clinical characteristics of RA and HC were shown in Supplementary Table 1.

Cell culture

Human rheumatoid fibroblast-like synoviocytes MH7A cells used in this study were a generous gift from Dr. Seiichi Tanuma (Tokyo University of Science). MH7A cells were isolated from the intra-articular soft tissue of knee joints of RA patients and were established as a cell line by transfection with the SV40 T antigen. Primary RA FLSs samples were obtained from synovium biopsy and OA FLSs were derived from arthroplasty. Written consent was signed by these patients. MH7A and RA/OA-FLSs cells were respectively cultured in 1640 and DMEM medium supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) 100 U/mL penicillin and 100 ug/mL streptomycin (Sigma Aldrich, St. Louis, MO, USA) at 37°C in a humidified atmosphere of 5% CO₂ in air.

Plasmid construction and luciferase reporter assay

MiR-15a/16 mimics and control were purchased from GenePharma (Shanghai, China). SOX5 3’UTR region was amplified from 293T cell genomic DNA, and the PCR product was cloned into pmiR-RB-ReportTM dual-luciferase reporter vector (Ribobio, Guangzhou, China) to generate SOX5 3’UTR wild-type (WT) and mutant plasmids. 293T cells were co-transfected with luciferase reporter constructs and WT or mutated corresponding SOX5 using Lipofectamine 2000 DNA Transfection Reagent (Thermo Fisher Scientific, Cleveland, OH, USA), according to the manufacturer's protocol. After transfection for 48 hours, the activity of luciferase constructs was measured using a DualGlo Luciferase Assay System (Promega) according to the manufacturer’s instructions. The firefly luciferase activity was used for normalization. For construction of SOX5 overexpression vector, the full-length of SOX5 cDNA was generated via standard PCR and inserted into the pAd- CMV-MC5 as described (Ad-SOX5) [4].

Cell proliferation, migration and invasion assay

MH7A cells were plated at a density of 8 × 10³ cells/well in a 96-well culture plate. miR-15a/16 mimics and control were transfected into MH7A using Lipofectamine 2000. The viability of cells was assessed by the Cell Counting Kit-8 (CCK-8; Dojindo China Co., Ltd., Shanghai) after transfecting for 24h, 48h and 72h, respectively. After transfecting with miR-15a/16 mimics and control for 48h, 5×10⁴ MH7A cells were placed into the chamber (8μm pore size, Corning, NY) with or without pre-coated with 50ul 1:5 diluted Matrigel (BD, San Jose, CA, USA). The transfected cells were resuspended in serum-free media and seed on the upper chamber for 24h, and the lower chamber were filled with complete media. Cells transmigrated into lower wells were fixed and stained with 0.1% crystal violet. The number of migrated cells was averaged from five 10×field-of-view images and normalized to control.

Immunofluorescent staining for F-actin

Transfected FLSs were resuspended and seeded into 35mm confocal dish for 24h. Dishes were washed with PBS and fixed with 4 % paraformaldehyde in PBS for 20 mins. To observe F-actin, cells were stained with 5ug/ml FITA- phalloidin (Sigma, St. Louis, MO, USA). Nuclei were co-stained with 6-diamidine-20-phenylindole dihydrochloride (DAPI) (Sigma, St. Louis, MO, USA). Stained cells were examined and photographed using a laser scanning confocal microscope.

Real-time quantitative PCR (qPCR)

For miRNAs expression, RNAs were extracted from serums using mirVanaTM Protein and RNA Isolation System (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. miRNAs were reverse transcribed using specific stem-loop miRNAs primers. About 300ng of RNA was used for reverse transcription. Small nuclear RNA U6 was used for normalization. RT-qPCR was performed using an Applied Biosystems 7900HT Fast Real-Time PCR System. For mRNA expression, total RNAs were extracted from primary FLSs or transfected cells using Trizol agent (Invitrogen) according to the manufacturer’s instructions. Total RNA was converted to cDNA using the Superscript II reverse transcriptase (Invitrogen). Primers used to amplify interest genes were listed in Supplementary Table 2. The measurement of specific gene expression was performed using the ABI Prism 7900 Sequence Detection System and analyzed using the ABI Prism 7900 SDS Version 1.0 software. Relative expressions of interest genes were normalized to β-actin and calculated by the 2^-ΔΔCt method.

Western blotting assay

Western blotting was performed using whole cell lysates. Non-specific interactions were blocked with 5% skim milk for 2 hours and were then probed with phospho-p38, phospho-ERK, and phospho-JNK and then were re-probed with antibody against total p38, ERK, JNK (Cell Signaling Technology, Danvers, MA, USA) and SOX5 (Abcam, Cambridge, MA, USA). β-actin (Cell Signaling Technology, Danvers, MA, USA) was used as a protein loading control. Protein bands were visualized with Super Signal West Dura chemiluminescent detection reagents following the manufacturer's directions.

Statistical analyses

All experiments were performed in triplicate. Results were expressed as the mean ± standard deviation (SD). The statistical significance of difference between an experimental group and its corresponding control was evaluated by a non-parametric Mann-Whitney U test (GraphPad Prism 8). The correlation between the relative expression levels of miR-15a/16 and SOX5 was analyzed using Spearman’s ρ-test. p < 0.05 was considered statistically significant.