MicroRNA-17 Suppresses TNF-α Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts

Abstract

TNF-α is a major cytokine implicated in rheumatoid arthritis (RA). TNF-α expression is shown to be regulated at transcriptional as well as posttranscriptional levels. However, the impact of changes in microRNA (miRNA) expression on posttranslational processes involved in TNF-α signaling networks is not well-defined in RA. Here we evaluated the effect of miR-17, a member of miR-17-92~ cluster, on TNF-α signaling pathway in human RA synovial fibroblasts (RASFs). We demonstrated that miR-17 expression was significantly low in RA serum, SFs, and synovial tissues as well as in the serum and joints of adjuvant-induced arthritis rats. RNA sequencing analysis showed modulation of 664 genes by pre-miR-17 in human RASFs. Ingenuity pathway analysis of RNA sequencing data identified the ubiquitin proteasome system (UPS) in TNF-α signaling pathway as a primary target of miR-17. Western blot analysis confirmed the reduction of TRAF2, cIAP1, cIAP2, USP2, and PSMD13 expression by miR-17 in TNF-α-stimulated RASFs. Immunoprecipitation assays showed that miR-17 restoration increased the K48-linked polyubiquitination of TRAF2, cIAP1, and cIAP2 in TNF-α-stimulated RASFs. Thus, destabilization of TRAF2 by miR-17 reduced the ability of TRAF2 to associate with cIAP2, thereby resulting in the downregulation of TNF-α-induced NF-κBp65, c-Jun, and STAT3 nuclear translocation and the production of IL-6, IL-8, MMP-1, and MMP-13 in human RASFs. In conclusion, this study provides evidence for the role of miR-17 as a negative regulator of TNF-α signaling by modulating the protein ubiquitin processes in RASFs.

MicroRNAs (miRNAs) are highly conserved set of single stranded non-coding RNAs (~19-23-nucleotide length) that are important in many developmental and physiological processes (1, 2), and their aberrant expression has been correlated with inflammatory diseases, including rheumatoid arthritis (RA) (2-5). A key specificity determinant for miRNA target recognition is based on Watson-Crick pairing of 5’-proximal “seed” region (nucleotide 2 to 8) in the miRNA to the seed match site in the target mRNA, which positioned mostly in 3’UTR (6) with a small subset of miRNAs targeting mRNA 5’UTR and/or coding region (7-9). While the exogenous delivery of different miRNAs has been shown to regulate various target genes in specific cellular context, the predicted impact of changes in miRNA expression on cellular processes and cytokine signaling networks is difficult to predict. Recent studies have shown significant changes in the expression of many genes by individual miRNAs overexpression (10-12). However, only a portion of differentially regulated genes were predicted direct target indicating that most of the changes in gene expression induced by miRNA transfections are indirect (12, 13).

Ubiquitination is a posttranslational modification process which plays a key role in various signal transduction cascade by ‘priming’ signaling proteins for either degradation or stabilization through Lys-linked K48 or K63 ubiquitin chains, respectively (14). The ubiquitin proteasome system (UPS) consists of ubiquitin ligases (1, 2, and 3) and proteome that mediates posttranslational modifications in the cytokine or toll-like receptors (TLRs) signaling network. For instance, upon binding to TNF-α, TNFR1 recruits adapter proteins, the protein kinase RIP1, several ubiquitin E3 ligases such as TRAF2, cIAP1, cIAP2 and the de-ubiquitination (DUB) enzymes for downstream signaling (15). However, TNF-dependent recruitment of multiple ubiquitin ligases and DUB enzymes implies the importance of ubiquitination for regulating inflammation and cell death in this pathway. However, being two different spectrums of biological processes, namely epigenetics and posttranslational, the influence of miRNAs on the ubiquitination of TNF-α signaling proteins is not well known in RA.

MiR-17~92 is located in the locus of MIR17HG (miR-17-92 cluster host gene), also known as C13orf25 (chromosome 13 open reading frame25). The miR-17-92 cluster transcript spans 800 nts and encodes six miRNAs that are transcribed from the same promotor (Supplementary Fig. 1A). However, these six miRNAs can be grouped into four families, based on their seed regions: miR-17, miR-18, miR-19 and miR-92. MiR-17 and miR-19 families are composed of the pairs of miRNAs with identical seed regions: miR-19/miR-20a and miR-19a/miR-19b-1 (16). As oncomirs, these miRNAs are known to promote proliferation, inhibit apoptosis, and induce tumor angiogenesis (17, 18). Yet in some context, the miR-17 family has been shown to negatively regulate cell proliferation (19-21) and inhibits cell migration and invasion in cancer (22, 23). Recent studies showed that miR-20a from the same cluster regulates apoptosis signaling kinase (ASK)1, whereas miR-19a/b were shown to regulate IL-6 and MMP-3 expression in lipopolysaccharides (LPS) activated RASFs (24, 25). In contrast, TNF-α- induced miR-18a has been reported to facilitate cartilage destruction and chronic inflammation in the joint through a positive feedback loop in NF-κB signaling, with a concomitant up-regulation of MMPs and mediators of inflammation in RASFs (26), suggesting differential effect of miRNAs from this cluster. In the present study, contrary to the other miRNAs of the same cluster, only miR-17 expression was consistently low in RASTs and RASFs, but not in OASTs or OASFs, making miR-17 more disease relevant. Thus, this study was undertaken to determine the role of miR-17 in RA pathogenesis.

Our results in the present study showed that miR-17 is consistently low in the diseased serum, STs, SFs, and in a rat adjuvant-induced arthritis (AIA) model of RA. To further extend these findings, the present study was carried out to study the effect of miR-17 overexpression on the posttranslational ubiquitination in TNF-α signaling. The results of this study showed that miR-17 overexpression inhibited TRAF2 expression and its association with cIAP2, thereby suppressing TNF-α signaling pathways and downstream inflammatory proteins. This study provides a novel insight to the role of miR-17 in down-regulating TNF-α signaling by influencing the protein ubiquitination pathway in RASFs.

Materials and Methods

Reagents and Antibodies

Rabbit polyclonal anti-TRAF2 (#sc-876), mouse monoclonal β-actin (#sc-47778), mouse monoclonal anti-USP-14 (#sc-100630), mouse monoclonal anti-MMP-1 (#sc-58377), rabbit polyclonal anti-MMP-13 (#sc-30073), rabbit polyclonal anti-Lamin A/C (#sc-20681) and rabbit polyclonal anti-p-IkB-α (#sc-8404) antibodies were purchased from Santa Cruz Biotech, (Santa Cruz, CA). Rabbit monoclonal Anti-cIAP1 (#7065), rabbit monoclonal anti-cIAP2 (#3031), rabbit anti-USP2 (#8036), rabbit monoclonal anti-RAD23A (#24555), anti-K63 polyubiquitin (#5621), and anti-K48 polyubiquitin (#8081), anti-STAT-3 (#9132), anti-NF-κBp65 (#8242), anti-p-c-Jun (S73) (#9164), anti-p-p38 (T180/Y182) (#4511), anti-p-JNK (T183/Y185) (#9251), total JNK (#8690), total p-38 (# 9258) and p-STAT-3 (S727) (#9134) antibodies were purchased from Cell Signaling Technology (Beverly, MA). TRAF2 mouse monoclonal (#AM1895B) for immunoprecipitation were purchased from Abjent (San Diego, CA). Anti-PSMD13 (#5937-1) antibody was purchased from Epitomics (Burlingame, CA), respectively. Total ASK1 (#ab131506), p-ASK1 Thr838/845 antibodies were was purchased from Abcam (Cambridge, MA) and Cell Signaling Technology (Beverly, MA), respectively. Human Cytokine Array C5 (#AAH-CYT-5) was purchased from Ray Biotech (Norcross, GA). SMARTpool ON-TARGET plus ASK1 siRNA or negative control siRNA was purchased from GE Dharmacon (Lafayette, CO).

Isolation and culture of human healthy (NL), osteoarthritis (OA), and RASFs

The procurement of de-identified human healthy/non-diseased (NL)-, OA-, and RA synovial tissue (ST) was done under the Institutional Review Board (IRB#106628) approved protocol. Human SFs were derived from synovial tissue of patients diagnosed with OA and RA from autopsies/amputation who had underwent total joint replacement surgery (mostly knee joints) or synovectomy. NL STs from non-arthritis individuals were obtained at the time of autopsy or amputation. Synovial tissue from 18 RA (Mean age ± SD; 74.2 ± 8.5), 12 OA (Mean age ± SD; 73.8 ± 12.7), and 8 NL (Mean age ± SD; 60.5 ± 9.8) were used in the present study. The de-identified human healthy/non-diseased (NL)-, OA-, and RA synovial tissue (ST) tissues were obtained from Cooperative Human Tissue Network (CTHN;, Columbus, OH) and National Disease Research Interchange (NDRI; Philadelphia, PA). Tissue specimens were washed by sterile PBS, minced, and processed as previously described (27). SFs were grown in RPMI 1640 containing 2 mM L-glutamine with 10% fetal bovine serum (FBS), at 37 °C, in a humidified atmosphere with 5% CO₂. Cells were used between passages 5 and 10 for these studies. For some studies, RNA was directly prepared from ST from NL donors, OA and RA patients or rat AIA or naïve joints.

Treatments of SF and preparation of microRNAs

NL, OA, or RA SFs were plated in 60 mm dishes and used when >80% confluent. SFs were serum starved overnight and then stimulated with or without TNF-α (20 ng/ml) for indicated time and cell lysates were prepared. Human STs and the joint homogenates from AIA study were also grounded to a fine powder in liquid nitrogen using a tissue pulverizer. Pulverized tissue was used to purify total RNA containing miRNA fraction (miRNeasy kit, Qiagen, Valencia, CA) to study miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92 expression.

Transient Transfection

RASFs or THP-1 differentiated macrophages were transfected with pre-miR-17 (Life Technologies, Carlsbad, CA) in 6-well plates or 100mm dishes or 150 mm dishes. RASFs were transfected with pre-miRNAs (100 nM) of miR-17 with negative control pre-miRNAs (Life Technologies) or anti-miR-17 (150 nM) with negative control a nti-miR using Lipofectamine® RNAiMAX transfection reagent (Life Technologies) for 48 h and then stimulated with or without TNF-α (20 ng/ml) for 30 min or 24 h. Total RNA containing miRNA fraction or cell lysate were prepared. Protein expression were determined using Western immunoblotting, respectively. Transfection efficiency was confirmed by the significant up regulation of miR-17 expression using TaqMan assays (Life Technologies). THP-1 cells were differentiated into macrophages by the treatment with Phorbol 12-myristate 13-acetate (PMA; 300 ng/ml) for 3 h and then transfected with pre-miRNAs (100 nM) or negative control using Lipofectamine® RNAiMAX transfection reagent (Life Technologies) for 48 h followed by treatment with or without TNF-α (20 ng/ml) for 30 min. RASFs (n=4) were also transfected with ASK1 siRNA or Negative control siRNA for 48 h using Lipofectamine® RNAiMAX transfection reagent (Life Technologies) for 48 h and then stimulated with or without TNF-α (20 ng/ml) for 24 h. RASFs were pretreated with the selective inhibitor of ubiquitin-conjugating enzyme E1 (PYR41; 1μM) for 2 h and then transfected with pre-miR-17 or NC-pre-miR for 48 h followed by 24 h stimulation with TNF-α.

Quantitative Real Time-PCR analysis

Total RNA was reverse-transcribed using SuperScript™ First Strand cDNA synthesis kit (Life Technologies) according to the manufacturer's protocol. Expression of miR-17 (also known as miR-17-5p), miR-18a, miR-19a, miR-19b, miR-20a, and miR-92-1 was quantified using TaqMan microRNA Assays with U6snRNA as control (Life Technologies). Expression of ASK1 mRNA was quantified using SYBR green qRT-PCR and GAPDH was used as control. Quantification of the relative expression was done by ΔΔC_t method.

RNA isolation, reverse transcription, and miR-17 qRT-PCR in serum samples

Exiqon serum RNA purification protocol was followed for the total RNA containing small RNA fraction using miRCURY™ RNA Isolation kit-Biofluids kit (Exiqon, Woburn, MA). Serum samples from healthy and RA donors, AIA rats and naïve controls were thawed on ice. Two hundred microliter of human serum or 50 μl of rat serum from each donor was transferred in 1.5 ml Eppendorf tube and centrifuged at 3,000 x g for 5 min at 4 °C to remove debris. Serum was transferred into new 1.5 ml Eppendorf tube and 60 μl of lysis buffer was added containing 1.17 μl of carrier RNA (0.8 μg/μl) from bacteriophage MS2. Samples were incubated at room temperature for 3 min and subsequently mixed with 20 μl of protein precipitation solution. After centrifugation at 11,000 x g, the aqueous phase containing the RNA was carefully transferred into a new collection tube, and RNA was precipitated with isopropanol. The mixture was applied to miRNA Mini spin column and washed several times, and RNA was eluted by the addition of 50 μl of RNase free water. Extracted RNA was used the same day for cDNA synthesis using the Universal cDNA synthesis kit II and UniSp6 RNA spike-in control primer. Quantitative PCR was performed in 10 μl reactions containing 4 μl of 40X diluted RT product, 5 μl of 2X SYBR^® green master mix, and 1 μl of UniRT LNA PCR primers’ for miR-17. Reaction mixtures were incubated at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 sec and 60 °C for 1 min followed by melting curve stage. miRNA-93-5p was used as reference control for sample analysis (28). A threshold cycle (Ct) was observed in exponential phases of the amplification and quantification of the relative expression levels was determined by the ΔΔCt method.

Bioinformatics analysis

Ingenuity Pathways Analysis (IPA) was used to interpret the differentially expressed genes in terms of an interaction network that might be altered as a result of RNA changes induced by miR-17 overexpression in RASFs compared to negative control. Genes with the P<0.05 (Student ‘t’ test) were selected from mRNA Sequencing data and a list of differentially regulated genes, and their corresponding expression values were uploaded to the IPA application software (Ingenuity® Systems, www.ingenuity.com). IPA was done with standard settings (Ingenuity knowledge case (gene only), direct and indirect, includes endogenous chemicals, consider only relationships where confidence =experimentally observed). Target ScanS 7.0, PicTar and miRanda algorithms were used to identify miR-17 binding sites in TRAF2, cIAP1 (BIRC2), cIAP2 (BIRC3), USP2 and PSMD13 mRNA.

RNA library preparation and sequencing

Human RASFs from two RA patients were transfected with pre-miR-17 or NC-pre-miR for 48 h and total RNA was prepared using miRNeasy kit (Qiagen). Next, total RNA integrity was checked using an Agilent Technologies 2100 Bio analyzer (Santa Clara, CA). 10 ng of high quality RNA was used to make cDNA for amplification with the Ion AmpliSeq Transcriptome Human Gene Expression kit (ThermoFisher Scientific, Grand Island, NY). The cDNA was subjected to 12 cycles of amplification with panel primers and barcoded with adapters as recommended. Resulting sequencing libraries were quantified by qPCR using SYBR FAST master mix from KapaBiosystems (Wilmington, MA). Sets of eight libraries were balanced, pooled and sequencing beads produced on an Ion Chef. Sequencing was performed on an Ion P1 semi-conductor sequencing chip using an Ion Proton™ System (ThermoFisher Scientific, Grand Island, NY). Data was collected and primary analysis performed using Torrent Suite software version 5.0.3. Reads were mapped to the panel and expression values determined. R Software version R-3.2.3 was used to generate heatmap (29). Microarray data have been deposited in a NCBI GEO database (accession number: GSE83930) and website address of the database is: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=ahgtwusgflmxnah&acc=GSE83930

Luciferase reporter assay

Luciferase reporter construct encompassing the 3’UTR of ASK1 mRNA (NM_005923) with the binding sites for miR-17 and empty control reporter construct were obtained from Genecopia (Rockville, MD). ASK1 3' UTR sequences was inserted downstream of the secreted Gaussia luciferase (GLuc) reporter gene driven by SV40 promoter. A secreted Alkaline Phosphatase (SEAP) reporter driven by a CMV promoter cloned into the same vector served as the internal control for normalization. RASFs were co-transfected with ASK1 luciferase reporter construct (1 μg) with pre-miRNA (100 nM) of miR-17 or negative control pre-miRNA (100 nM) (Life Technologies, CA, USA) using Lipofectamine® 2000 transfection reagent (Life Technologies). After 48 h, the condition media was collected and luciferase activity was assayed using a Secrete-Pair™ Dual Luminescence Assay kit (Genecopia, Rockville, MD). Each experiment was repeated in three independent SF donors and each assay was performed in triplicate.

Preparation of nuclear extracts

To study the effect of miR-17 on TNF-α-induced NF-κBp65, p-c-Jun and p-STAT-3 activation, RASFs were transfected with pre-miR-17 or NC-pre-miR (100 nM) and then stimulated with or without TNF-α (20 ng/ml) for 30 min or 24 h. Upon termination, cells were washed with ice-cold PBS, collected by scraping, and centrifuged at 1,500g for 5 minutes at 4°C. Nuclear fractions were prepared and equal amount of protein (15 μg) from nuclear fractions was evaluated by Western blotting in order to study the level of NF-κBp65, p-c-Jun and p-STAT-3 expression using Western immunoblotting.

Western immunoblotting

SFs were lysed in RIPA lysis buffer with complete protease inhibitor cocktail (Roche, Indianapolis, IN). Cell lysates/tissue homogenates/nuclear fractions were resolved on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked with 5% non-fat dry milk powder/BSA in Tris buffered saline containing 0.1% Tween-20 (TBS-T), and probed with 1:1000 diluted polyclonal or monoclonal antibodies specific for TRAF2, cIAP-1, c-IAP-2, USP2, RAD23A, USP14, PSMD13, K63, K48, ASK1, STAT3, p-c-Jun, p-IκB-α, NF-κBp65, p-p38, p-JNK, total p38, total JNK, MMP-1, MMP-13 or β-actin. Culture supernatants from miR-17 transfected and TNF-α stimulated RASFs were concentrated and used for MMP-1 and MMP-13 expression. Immunoreactive bands were visualized using the HRP-linked secondary antibodies and enhanced chemiluminescence (BIORAD Molecular Imager ChemiDoc™ XRS+, Hercules, CA). Images were analyzed using the GELDOC software. Each band was scanned using Image lab 5.1 software and the expression values (pixels/band) were presented as Mean ± SE.

Enzyme-linked immunosorbent assays (ELISA)

Measurement of IL-6 and IL-8 in the culture supernatants of RASFs were analyzed using ELISA according to the instruction of manufacturer (R&D Systems).

Cytokines antibody array

We used a commercial cytokine antibody-based array designed to detect 80 different cytokines (RayBio Human Cytokine Array C5, RayBiotech, Norcross, GA). RASFs were transfected with pre-miR-17 or NC-pre-miR for 48 h followed by TNF-α-stimulation for 24 h. Condition media from four patients was pooled for each experimental condition and used in the assay. Experiments were performed essentially as recommended by the manufacturer. Briefly, array membranes were incubated for 1 h in 2 ml blocking buffer, then incubated overnight with 2 ml of the pooled (n=4; 500 μl from each) culture supernatant and washed. After incubation was over, a cocktail of 80 biotinylated antibodies diluted 1:250 was added to each membrane at 1 ml per array membrane and incubated for 5 h at 4°C overnight. Membranes were washed and sandwiched antigens were detected by the enhanced chemiluminescence method by incubating the membranes for 2 h with 2 ml of a peroxidase-labeled streptavidin solution (diluted 1:1000) and signals were captured on X-ray films. Arrays were processed simultaneously for image acquisition. Each spot intensities were analyzed using GELDOC software and the mean value was used. Normalized values were calculated by the formula: X (Ny) = X(y) * P1/P(y). Where P1 was average signal density of the positive control spot on the reference array, P(y) is an average signal density of the positive control spot on array “y”, X(y) is the signal density for a particular spot on array for sample “y”, and X (Ny) is the normalized value of a particular spot “X” on array for sample “y”.

Immunoprecipitation Assay

RASFs were transfected with pre-miR-17 or NC-pre-miR in 150 mm dishes for 48h, starved overnight followed by TNF-α stimulation for 30 min. Cells were washed 2 times in ice cold 1X PBS, lysed in 500 μl of RIPA buffer as described earlier, utilized for immunoprecipitation assays. Clear lysate were subjected to protein estimation using Bio-Rad DC method. Three microgram antibody was used per milligram whole cell extract (1 mg) from each sample was subjected for immunoprecipitation using TRAF2 (Abgent), K63-ubiquitin (Cell Signaling Technologies), or K48-ubiqutin (Cell Signaling Technologies) antibodies. A similar amount of nonspecific IgG control antibody (Flag M2, Sigma) was used as isotype control. Antibody and whole cell extract were incubated at 4°C on a rotor for overnight, followed by the incubation with protein G Sepharose beads for 4 h to capture antibody and protein complex. Beads were then subjected to 3 washes with RIPA wash buffer followed by final wash with 1X PBS. Protein beads complex were eluted by boiling in 2X SDS sample buffer and then resolved on 4-15% Bio-Rad TGX gel using Western immunoblotting. For K63 and K48 IP assays, 120 μl bed volume equivalent of Dynabeads (Invitrogen, Thermo Scientific) were subjected to crosslinking with 18 μl of K63 polyubiquitin antibody or 9 μl of K-48 polyubiquitin antibody (Cell Signaling Tech) using BS3 cross linker (Thermo Scientific) as per manufacturers’ instructions. Beads were equally divided in 3 samples for IP assay. Protein complex was eluted form cross-linked beads using 2X sample buffer for 15 min at 70 °C.

Induction of adjuvant-induced arthritis (AIA) in rats

Female Lewis rats (Harlan Laboratories, Indianapolis, IN), weighing 135-160 gm were injected subcutaneously at the base of the tail with 300 μl (5 mg/ml) of lyophilized Mycobacterium butyricum (Difco laboratories, Detroit, MI) in sterile mineral oil. The day of adjuvant injection was considered day 0. Body weight and ankle circumferences were measured on day 0, 8 and 18 in a blinded manner, as described previously (30). The study also included a naïve (no adjuvant) group for comparison. Naïve and AIA rats on day 8 and 18 were sacrificed. The ankle circumferences of both hind ankles from each rat were averaged, and ‘n’ is the number of rats used in each experimental group. On day 18, joints and serum samples of rats were collected. Animal study was approved by the IACUC committee of the Washington State University.

Statistical analyses

Student ‘t’ test (unpaired two tailed t-test) followed by one way ANOVA was used to calculate statistical differences between the results of the different variables. Values shown are Mean ± SE unless stated otherwise. Comparisons were performed using GraphPad Prism 6 software package and P<0.05 was considered significant.

Results

MiR-17 expression inversely correlates with RA pathogenesis

MiR-17~92 is a polycistronic miRNA cluster located in the chromosome 13 (Supplementary Fig. 1A). To identify the basal expression levels of miR-17~92-1 cluster in RA, we performed qRTPCR using RNA from SF and ST of healthy (NL) donors, OA and RA patients. The expression levels of all six miRNAs; miR-17 (~69%; P<0.01), miR-18a (~81%; P<0.05), miR-19a (~87%;P<0.01), miR-20a (~85%; P<0.01), miR-19b (~79%; P<0.01) and miR-92-1 (~80%; P<0.05) were significantly decreased in RA ST compared to NL ST (Supplementary Fig. 1B). Interestingly, we observed that miR-17 levels were consistently low in both ST and SF from RA donors compared to NL and OA donor ST and SF (Fig. 1A and B). To further verify miR-17 expression, we utilized a rat AIA model in which inflammation starts around day 8 and peaks around day 18 (31). Transcriptional analysis of pulverized joint tissue from naïve and AIA rats also showed a significant decrease in the expression of miR-17 on day 18 compared to the naïve group (Fig. 1C; P<0.05).

FIGURE 1.

Clinical significance of miR-17 expression levels in Rheumatoid Arthritis patients. (A) Expression profile of miR-17 in synovial tissue (ST) and (B) in synovial fibroblasts (SFs) obtained from NL, OA, and RA donors. (C) Expression levels of miR-17 in AIA (day 18) model of human RA compared to the naïve group. miRNA expression levels were evaluated using TaqMan based qRT-PCR assay. The values are represented as mean ± SEM for indicated number of animals. (A-C) U6snRNA was used as a reference control. (D) Lower miR-17 levels in the serum of human RA patients. miR-93-5p was used as a reference control. (E) AIA rats showing increase in ankle circumferences on day 18. (F) Lower miR-17 expression was associated with RA in these AIA rats. Expression of miR-17 was determined using qRT-PCR. miR-93-5p was used as a reference control. The values are represented as mean ± SEM for the indicated number of serum donors or animals used per group. * p<0.05; **p<0.01.

Furthermore, we observed a significant decrease in miR-17 serum levels in RA patients (Fig. 1D; P<0.05). To further validate these findings, we determined miR-17 levels in the serum of AIA animals and compared with the naïve group. AIA animals showed the signs of severe arthritis on day 18 as evident from a significant increase in ankle circumferences by ~65% when compared to the naïve group (Fig. 1E; P<0.01). A lower serum miR-17 level with the increase in ankle circumferences was observed in AIA rats on day 18 (Fig. 1F; P<0.01).

Protein ubiquitin pathway genes in RASFs are modulated by miR-17: confirmation by IPA analysis

To determine the role of miR-17 in RA, we used a gain-of-function model and performed RNA-sequencing analysis in miR-17 overexpressed RASFs. Among the panel of 20,803 genes, the expression of 15,067 genes as shown in the representative heat map was observed in pre-miR-17 and NC-pre-miR transfected RASFs (Supplementary Fig. 2A). A total of 664 significantly modulated genes (301 upregulated and 363 downregulated) were further utilized for the IPA analysis. The result of IPA predicted the protein ubiquitin pathway as a major canonical pathway affected by the differentially regulated genes (Supplementary Fig. 2B). Interestingly, IPA analysis generated an interactome that showed connectivity among various ubiquitin ligases, NF-ԟB family, AP-1/cJun, 20S and 26S proteasome system (Supplementary Fig. 2C).

The heat map generated based on the list of identified ubiquitin ligases and other proteasome pathway proteins regulated by miR-17 overexpression showed some of the key ubiquitin and de-ubiquitin ligases in TNF-α-signaling such as TRAF2, cIAP2, USP2, PSMD13, and RAD23A (Fig. 2A and B). This suggests that miR-17 may closely be involved in the regulation of ubiquitin proteasome system (UPS) in TNF-α-signaling. The IPA results confirmed that miR-17 overexpression may significantly influence canonical pathways with high pathological relevance, including protein ubiquitin pathway, TNF-α related weak inducer of apoptosis (TWAEK) and TNF-R1 signaling (Supplementary Fig. 3A). Functional network and canonical pathway analysis of thirty selected genes associated with TNF-α-signaling and ubiquitination is shown in Fig. 3B and Supplementary Fig. 4A-C.

FIGURE 2.

MiR-17 regulates genes related to TNF-α signaling in RASFs. (A) Heatmap pattern of the gene expression data for selected genes related to TNF-α signaling and ubiquitination. Each row represents a single transcript, and each column represents a single sample. Red represents lower expression; green, higher expression. (B) Fold change in the selected gene from RNA Seq data are presented as Mean ± SD (n=2) in a tabulated form. (C) Effect of miR-17 on the expression of selected genes (TRAF2, cIAP1, cIAP2, USP2, PSMD13, USP14 and RAD23A) was verified using qRT-PCR. RASFs (n=4) were transfected with pre-miR-17 or NC-pre-miR for 48 h and total RNA was prepared. GAPDH was used as a reference control. (D-E) Verification of RNA sequencing data for TRAF-2, cIAP1, cIAP2, USP2, USP14, PSMD13 and RAD23A (n=4) was performed using Western Immunoblotting. RASFs were transfected with pre-miR-17 or NC-pre-miR for 48 h and cell lysates were prepared. Densitometry for the Western blots for the indicated number of patients was performed and values were normalized with β-actin.

FIGURE 3.

MiR-17 regulates ubiquitination and de-ubiquitination process to inhibit TNF-α-signaling in RASFs. (A and B) Exogenous delivery of miR-17 and miR-20a in RASFs (n=4) showing a significant increase in the expression of both miRNAs after 48 h of transfection. (C) Effect of miR-17 on TRAF2, cIAP1, cIAP2, USP2, USP-14, PSMD13, and RAD23A in TNF-α-stimulated RASFs. RASFs were transfected with miR-17 for 48 h followed by stimulation with TNF-α for 24h. Cell lysates (n=4) were prepared and assayed for their expression by Western immunoblotting. Densitometry for the Western blots for the indicated number of patients was performed and values were normalized with β-actin. (D) RASFs were transfected with pre-miR-17/pre-miR-20a/NC-pre-miR for 48 h followed by TNF-α-stimulation for 30 min. Total cell lysates (n=3) were used to determine the effect on global expression of K63 (upper panel) and K48-linked (lower panel) polyubiquitination. (E and F) Figures show the densitometry analysis for K63 and K48 band intensity values that were normalized with β-actin. (G) RASFs (n=3) transfected with pre-miR-17/pre-miR-20a/NC-pre-miR for 48 h followed by 30 min TNF-α stimulation were immunoprecipitated with K63 polyubiquitin, or (H) K48 polyubiquitin and probed for the expression of TRAF2, cIAP1 and cIAP2.

We confirmed RNA sequencing results by qRT-PCR and Western blot analysis in miR-17 overexpressed RASFs, where our results showed a significant decrease in the basal expression level of TRAF2, cIAP1, cIAP2, USP2 and PSMD13 proteins, no change in the constitutive expression level of RAD23A and USP14 in miR-17 overexpressed RASFs (Fig. 2C-E).

MiR-17 restoration modulates E2 ligases to inhibit TNF-α-signaling in RASFs

To determine the impact of miR-17 on ubiquitination mechanisms essential for TNF-α signaling, RASFs were transiently transfected with miR-17 mimic or a negative control and stimulated with TNF-α for 24 h (Fig. 3). For comparison, we also included miR-20a, another miR-17 family member shown to have similar function (22, 32) Transfection with pre-miR-17 or miR-20a for 48 h showed a significant increase in the expression of both miRNAs compared to negative-control transfected samples (Fig. 3A and B). Consistent with our sequencing results, Western blot analysis showed that TRAF2, cIAP1, cIAP2, PSMD13, and USP2 expression was significantly reduced in TNF-α-stimulated RASFs transfected with pre-miR-17 compared to the negative control (Fig. 3C). However, the expression of RAD23A and USP14 was not altered by miR-17 overexpression in TNF-α-stimulated RASFs (Fig. 3C). To determine whether miR-17 is regulating the expression of TRAF2, cIAP1, cIAP2, USP2 and PSMD13 via the sequence spanning in the 3’UTR of their mRNA, we used bioinformatics algorithms (Target ScanS 7.0, PicTar and miRanda). No direct binding site of miR-17 in the 3’UTRs of TRAF2, cIAP1, cIAP2, PSMD13, or USP2 mRNA was observed (data not shown), which indicates that miR-17 may impact cellular processes in RASFs to regulate their gene expression.

To investigate the mechanism of TRAF2 degradation by miR-17 in response to TNF-α stimulation, we analyzed K63- and K48-linked ubiquitination pattern in miR-17 transfected RASFs. Surprisingly, our results showed that both miR-17 and miR-20a increased the global K63 ubiquitination in TNF-α-stimulated RASFs (Fig. 3D-E), which suggests miR-17 and miR-20a may be enhancing total K63 ubiquitination process to stabilize certain TNF-α signaling proteins. A modest decrease in the global K48-linked ubiquitination was also observed in TNF-α-stimulated RASFs, however this was not statistically significant (Fig. 3F).

To further examine the effect of miR-17 on K63 and K48 ubiquitination specific to TRAF2, cIAP1 and cIAP2 proteins, TNF-α-stimulated RASFs with or without control or miR-17 overexpression were immunoprecipitated with K63- and K48-linked ubiquitinated proteins from cell lysate and probed for TRAF2, cIAP1 and cIAP2 (Fig. 3G and H). However we did not observed significant changes in K63-mediated ubiquitination of TRAF2, cIAP1 and cIAP2 (Fig. 3G). In contrast, Western blotting results with K48-linked immunoprecipitated proteins showed that miR-17, as well as miR-20a, enhanced K48-mediated ubiquitination for TRAF2, cIAP1, and cIAP2 compared to the negative control in TNF-α-stimulated RASFs (Fig. 3H). These results suggest that miR-17 induces K48-mediated ubiquitination of key TNF-α signaling proteins TRAF2, cIAP1 and cIAP2 that may influence their stability and there by inhibits downstream signaling events in RASFs.

MiR-17 interferes with the association of TRAF2-cIAP2 in TNF-α-stimulated RASFs

To further understand the impact of miR-17 on the early events in TNF-α signaling, we treated RASFs with NC-pre-miR, pre-miR-17, and pre-miR-20a followed by TNF-α-stimulation for 30 minutes. Cell lysates were immunoprecipitated with TRAF2 antibody and analyzed for its association with the signaling partners cIAP1, cIAP2, and RAD23A in RASFs. We found that miR-17 preferentially inhibited the association of cIAP2 with TRAF2 in TNF-α-stimulated RASFs, but elicited no effect on RAD23A (Fig. 4A).

FIGURE 4.

MiR-17 interferes with the association of TRAF2-cIAP2 in TNF-α-stimulated RASFs. (A) RASFs (n=4) were transfected with pre-miR-17/pre-miR-20a/NC-pre-miR for 48 h followed by TNF-α-stimulation for 30 min. Cell lysates were immunoprecipitated with TRAF2 or IgG and probed for the changes in the expression of cIAP1, cIAP2, and RAD23A. Densitometric analysis for cIAP1 and cIAP2 band intensity is shown. (B) RASFs were transfected with miR-17 for 48 h followed by stimulation with TNF-α for 30 min. Cell lysates (n=4) were assayed for the expression of TRAF2, cIAP1, cIAP2, RAD23A and β-actin by Western immunoblotting. (C) THP-1 differentiated macrophages (n=3) were transfected with pre-miR-17/pre-miR-20a/NC-pre-miR for 48 h followed by TNF-α-stimulation for 30 min. total cell lysates were immunoprecipitated with TRAF2 or IgG and probed for the changes in the expression of cIAP1 and cIAP2. Densitometric analysis for cIAP1 and cIAP2 band intensity is shown. (D) THP-1 differentiated macrophages were transfected with miR-17 for 48 h followed by stimulation with TNF-α for 30 min. Cell lysates (n=3) were assayed for the expression of TRAF2, cIAP1, cIAP2 and β-actin by Western immunoblotting.

To investigate whether this effect of miR-17 is RASFs specific or not, THP-1 differentiated macrophages were transfected with NC-pre-miR, pre-miR-17, or pre-miR-20a followed by TNF-α for 30 min. Cell lysates were immunoprecipitated with TRAF2 and probed for the same panel of proteins mentioned in Fig. 4A and B. In agreement with findings in RASFs, we found a decrease in the association of TRAF2 with cIAP2 in macrophages (Fig. 4C). Similar to RASFs, no early impact of miR-17 overexpression was observed on the expression of these proteins as seen in the analyzed inputs (Fig. 4B and D). To study the impact of miR-17 on the ubiquitination pathways, a chemical inhibitor of ubiquitin-conjugating enzyme E1 (PYR41) with no effect on E2 or E3 ubiquitin ligases was used. RASFs were pretreated with PYR41 for 2 h and then transfected with miR-17 (Supplementary Fig. 2D and E). Our results showed that inhibiting the ubiquitin E1 ligase had no effect on TNF-α-induced IL-6 and IL-8 production, however, miR-17 was able to lessen this TNF-α-induced IL-6 and IL-8 production in RASFs (Supplementary Fig. 2D and E). These findings suggest that miR-17 may potentially interfere with the UPS that impacts the stability and efficiency of ubiquitin E3 ligase TRAF2 to associate with cIAP1/cIAP2 complex and participate in TNF-α signaling.

Impact of miR-17 on MAPK/NF-κB/ STAT3 pathways in TNF-α-stimulated RASFs

MiR-17 overexpressed RASFs were stimulated with TNF-α for 30 mins or 24 h to study regulation of signaling proteins and the soluble proteins, respectively. We observed that miR-17 overexpression led to a decrease in p-p38 and p-JNK expression in TNF-α-stimulated RASFs (Fig. 5A and B). Apoptosis signal-regulating kinase 1 (ASK1; a serine-threonine kinase) regulates downstream p38 and JNK pathways and has been shown to play a critical role in RA pathogenesis via TNF-α signaling (33, 34). Activation of ASK1 is tightly regulated by the phosphorylation of threonine residue (Thr₈₃₈ and Thr₈₄₅ of human and mouse ASK1, respectively) (33, 34). A higher expression of total ASK1 and ASK1 Thr₈₃₈ was found in RASFs compared to NL SFs (Fig. 6A-C). Interestingly, we found that miR-17 binds to ASK1 3’UTR to regulate its expression in RASFs, which may downregulates p-p38 and p-JNK (Fig. 6D-F). Interestingly, ASK1 knockdown also inhibited TNF-α-induced IL-6 and IL-8 production in RASFs (Fig. 6G). MiR-17 has been shown to target STAT3 3’UTR and regulate STAT3 expression, thereby, leading to a loss of suppressive function in myeloid-derived suppressive cells (35, 36). Consistent with the previous findings, an overexpression of miR-17 reduced the nuclear translocation of p-STAT3 in TNF-α-stimulated RASFs (Fig. 5A-B). Furthermore, we found that miR-17 also inhibited TNF-α-induced IκB-α phosphorylation when compared to negative control (Fig. 5A and B). Aligned with these findings, our results showed that miR-17 moderately reduced TNF-α-induced activation and nuclear translocation of transcription factors NF-κBp65 and p-c-Jun in RASFs (Fig. 5A and B).

FIGURE 5.

Impact of miR-17 on MAPK/NF-κB/STAT3 pathways and downstream mediators in TNF-α-stimulated RASFs. (A-B) Effect of miR-17 on the p38, JNK, IκB-α phosphorylation and nuclear translocation of NF-κBp65, p-c-Jun, p-STAT3 in TNF-α-stimulated RASFs. RASFs (n=3) were transfected with pre-miR-17 for 48 h and then stimulated with or without TNF-α for 30 min. Total cell lysates were probed for p38, JNK, I-ԟαBand and Nuclear fractions for NF-κB p65, p-c-Jun and p-STAT3 were prepared and assayed using Western immunoblotting. (B) Densitometric analysis for the Western blots (n=3) is shown. Band intensities were normalized with total their respective total forms, β-actin or Lamin A/C (for nuclear fraction). (C) Human cytokine antibody array (Ray Biotech Inc.) was used to measure the secretion of 80 cytokines in the pooled conditioned medium (n=4) from miR-17 or NC-miR overexpressed and TNF-α (20 ng/ml) 24 h stimulated RASFs. (D and E) RASFs (n=4) were transfected with pre-miR-17 for 48 h and then stimulated with TNF-α for 24 h to determine IL-6 and IL-8 production using quantitative ELISA. The values are represented as mean ± SEM for indicated number of SF donors. * p<0.05. (F-G) RASFs (n=4) were transfected with anti-miR-17 for 48 h and then stimulated with TNF-α for 24 h to determine IL-6 and IL-8 production using quantitative ELISA. The values are represented as mean ± SEM for indicated number of SF donors. * p<0.05. (H-I) RASFs were transfected with pre-miR-17 or negative control for 48 h, followed by TNF-α-stimulation for 24 h. Condition media (n=3) was concentrated using Amicon ® Ultra Centrifugal filters (Millipore) and analyzed for MMP-1 and MMP-13 protein expression using Western Immunoblotting. Densitometric analysis for the MMP-1 and MMP-13 (n=3) is shown.

FIGURE 6.

MiR-17 may directly target ASK1 3’UTR to regulate its expression in RASFs. (A-C) Expression of total ASK1 and p-ASK Thr₈₃₈ protein expression was evaluated in NL, OA, and RA SFs by Western immunoblotting. (D) Bioinformatics analysis for the predicted binding sites of miR-17 in ASK1 mRNA 3’UTR and their cross species conservation. (E) Inhibitory effect of miR-17 on ASK1 3’UTR luciferase reporter activity in RASFs. ASK1 reporter vectors was transfected in RASFs (n=3) with pre-miR-17 or NC-pre-miR. Experiment was performed in triplicate with indicated number of patients. (F) Effect of pre-miR-17 transfection on ASK1 mRNA and protein expression at the basal level and in TNF-α-stimulated RASFs (n=3) was studied using qRT-PCR and Western immunoblotting respectively. GAPDH or β-actin was used as a loading control. (G) Effect of ASK-1 siRNA on the TNF-α induced IL-6 and IL-8 production in RA SF was analyzed using ELISA. RA SFs (n=4) were transfected with ASK1 siRNA for 48 h and then stimulated with TNF-α for 24 h. Condition media was analyzed for IL-6 and IL-8 production. The values are represented as mean ± SEM for indicated number of patients using different donors. * p<0.05; **p<0.01. (H) An overview of the effects of miR-17 on TNF-α induced MAPK, NF-κB and STAT3 signaling in RASFs. Schematic representation of the effect of miR-17 on TNF-α-signaling proteins in RASFs. Findings of the present study suggest that miR-17 modulation of the UPS is an important mechanism to inhibit RASFs-mediated inflammation and tissue destruction in RA.

MiR-17 inhibits TNF-α-induced IL-6, IL-8 and MMP production in RASFs

To understand the impact of TNF-α signaling inhibition by miR-17, we determined the effect of miR-17 overexpression on the production of 80 different cytokines, chemokines, and growth factors using cytokine array. Densitometry analysis of the array membrane results obtained from the conditioned media of RASFs treated with NC-pre-miR or miR-17 in the presence of TNF-α showed a marked inhibition of several pro-inflammatory cytokines and chemokines known to contribute to RA pathogenesis including, G-CSF (~24%), GM-CSF (~19%), GRO (~17%), GRO-α (~31%), CCL23 (~21%), IL-3 (~16%), IL-6 (~15%), IL-7 (~26%), IL-8 (~19%), MCP-1 (~15%), M-CSF (~25%), MDC (~24%), MIG (~24%), MIP-1β (~25%) and TNF-β (~22%) (Fig. 5C). To confirm this array result, we used the same conditioned media to quantitate IL-6 and IL-8 production by ELISA method. Consistent with the array results, miR-17 overexpression inhibited TNF-α-induced IL-6 and IL-8 production in RASFs (Fig. 5D and E). In contrast, the inhibition of endogenous miR-17 expression showed a significant increase in IL-6 and IL-8 production in TNF-α-stimulated RASFs (Fig. 5F and G). Extending these findings, our results also show that overexpression of miR-17 inhibited TNF-α-induced MMP-1 and MMP-13 production in RASFs (Fig. 5H and I), suggesting a protective role of miR-17 in cartilage degradation.

Discussion

In the present study, we identified the role of miR-17 in modulating posttranslational protein ubiquitination pathway to down-regulate TNF-α-signaling events in RA (Fig. 6H). We identified that miR-17 expression is severely low in RA and its restoration in vitro may induce K48-linked ubiquitination of TRAF2 and cIAP2 and may reduce their association which results in the reduction of TNF-α-induced inflammatory mediators in RASFs. Importantly, this study validated the IPA data that suggests miR-17 may influence posttranslational modifications independent of its 3’UTR binding. These findings provide opportunity for further understanding of the impact of miRNA on cellular and posttranslational mechanisms such as protein ubiquitination pathway that are important in the cytokine signaling networks for RA pathogenesis.

TRAF2, an E3 ubiquitin ligase, is a critical upstream component in the TNF-α signaling that activates MAPK and NF-κB pathways through the recruitment of cIAP1 and cIAP2 to TNFR signaling complex (37, 38). TRAF2 is tightly regulated by posttranslational modifications such as auto-phosphorylation, ubiquitination or de-ubiquitination (39). Among these, ubiquitination plays an important role in diverse cellular events and signaling; however, this mechanism in pro-inflammatory cytokine signaling networks is not well studied for RASFs (40, 41). Our results showing that miR-17 reduced K48-linked ubiquitination in RASFs, further confirms the IPA findings of the influence on protein ubiquitin pathways. The UPS is indispensable for TNF-α signal transduction and balances the cellular expression and activity of proteins by ubiquitination and degradation (42). In contrast, DUBs remove ubiquitin conjugates by activating ATP hydrolysis, thereby, rescuing proteins from degradation (43). Among the different DUBs (USP14, USP2 and PSMD13) modulated by miR-17 in RNA sequencing data, our results in RASFs lysates confirmed that miR-17 suppressed the expression of PSMD13 and USP2 in TNF-α-stimulated RASFs. These results suggest that miR-17 affects stability of TRAF2 and/or cIAP2 proteins by partly regulating de-ubiquitination activity of PSMD13, an important proteome component, and USP2 an ubiquitin-specific protease important in TNF-α-induced RASFs. USP2 has been shown to target multiple substrates for protein stability e.g. p53, cyclin D1 (44) and its mRNA may be modulated by TNF-α in a cell-specific manner (45). Further investigation is needed to understand the mechanism involved in down-regulation of USP2 by miR-17. In agreement with our results, mir-17-92 cluster has previously been shown to targets E3 ubiquitin ligases, thereby affecting PTEN subcellular localization through monoubiquitination of limb-innervating lateral motor neurons (46).

Our findings from IP assays showed that miR-17 as well as miR-20a, enhanced K48-mediated ubiquitination for TRAF2, cIAP1, and cIAP2, suggesting that these miRNAs induce K48-mediated proteasomal degradation of key TNF-α signaling protein TRAF2, cIAP1 and cIAP2 to inhibit their expression and downstream signaling. Further validation studies in THP-1 activated macrophages also confirmed that indeed the proteasomal degradation of TRAF2 and cIAP2 by miR-17 further reduced their association and possibly downstream signaling events.

In a classical TNF-α signaling, TNF-R2 forms complex with TRAF2 and cIAP1/2 to further recruit and activate RIPK1, which further activates TAK1 kinase activity leading to the activation of NF-κB and MAPK pathways (15). Our results showed that due to lack of TRAF2/cIAP2 association and efficient signaling activation by miR-17, we observed an inhibition in the activation of TNF-α-induced p-p38, p-JNK, and p-IԟB-α and consequent reduction in the nuclear translocation of p-c-Jun and NF-κBp65. The inhibition of p-c-Jun and NF-κB transcription factors also caused a significant reduction in TNF-α-induced MMP-1 and MMP-13 production in RASFs. We have recently shown that a similar reduction in TRAF6 and TAK1 association by epigallocatechin-3gallate (EGCG) inhibited IL-1β signaling and downstream mediators of inflammation in RASFs (47). It is worth noting that both MMP-1 and MMP-13 have no 3’UTR binding regions for miR-17, which further suggest this to be an indirect regulatory effect on tissue remodeling mediated by RASFs Our results showed that miR-17 targets ASK1 3’UTR to regulate ASK1 expression, which may result in the reduction of p38 and JNK phosphorylation in RASFs. A decrease in TNF-α-induced IL-6 and IL-8 production was observed by miR-17 overexpression as well as by ASK1 knockdown in RASFs. Importantly, in addition to directly targeting ASK1, miR-17 may influence the association of TRAF2 and cIAP2 in TNF-α-stimulated RASFs. We hypothesize that miR-17 may regulate TNF-α signaling at multiple steps in RA and its influence on TRAF2 and cIAP2 may be the earliest and most prominent in TNF-α signaling pathway. In addition, recent studies have shown that miR-17 targets STAT3 3’UTR to regulate its expression in myeloid-derived suppressive cells and in other cell types (35, 36), suggesting STAT3 regulation may be a downstream direct effect of miR-17 through 3’UTR binding.

The miR-17–92 cluster has been shown to influence acquired and innate immune responses (48). However, individual members of this cluster have shown different biological activities. This is evident from our results where miR-17-mediated suppression of TRAF2/cIAP2 association. Furthermore, miR-17 has been shown promote osteogenic differentiation in the inflammatory microenvironment and miR-20a has promoted osteogenesis of hMSCs via BMP signaling (49, 50). In contrast, miR-18a enhanced MMP-1, IL-6 and IL-8 expression in TNF-α-stimulated RASFs (26). Another study showed the suppression of miR-19a and miR-19b in RASFs by TLR ligands and identified TLR2 as a direct target of miR-19 family (25). These findings suggest that individual miRNA in a cluster might have a completely different influence, depending on the level of expression and the cytokine signaling pathways involved that contribute to RA pathogenesis.

A key determinant for miRNA target recognition is the seed match site in the target mRNA, which positioned mostly in 3’UTR (6). In the present study, in silico prediction analysis showed no direct binding site of miR-17 in the 3’UTRs of TRAF2, cIAP1, cIAP2, PSMD13, and USP2 mRNA indicating that miR-17 may not target respective mRNA 3’UTRs of these genes to regulate their expression in RASFs. A recently described model to account for indirect effect of miRNA transfection postulates that the transfected miRNAs may compensate endogenous miRNAs for the available RISC and consequently alter the regulation of target of these endogenous miRNAs (51). Recent studies have shown that among all the genes that were differentially expressed with the endogenous miRNA modulation, less than 20% of those genes were predicted miRNA targets (12), suggesting the underlying coordinated changes in overall patterns of gene expression involve the modulation of centralized “hub genes” (52). These regulatory genes (i.e. hub genes) have the potential to control group of downstream genes to force differential gene expression outcomes (53). Our findings is an important step towards understanding the impact of miRNAs on the essential cellular processes and TNF-α signaling network in RASFs, a cell type that has not been targeted for therapeutic approaches. However, further studies are desired to extend these in vitro findings in animal models of human RA to understand the synovial and systemic benefit of therapeutically restoring miR-17 expression level in RA.