HHS Public Access Author manuscript Author Manuscript
J Immunol. Author manuscript; available in PMC 2017 September 15. Published in final edited form as: J Immunol. 2016 September 15; 197(6): 2219–2228. doi:10.4049/jimmunol.1600360.
MicroRNA-17 Suppresses TNF-α Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts Nahid Akhtar#, Anil Kumar Singh#, and Salahuddin Ahmed#,* #Department
Author Manuscript
of Pharmaceutical Sciences, Washington State University College of Pharmacy, Spokane, 99204, Washington, United States
Abstract
Author Manuscript
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.
Keywords
Author Manuscript
Rheumatoid Arthritis; microRNA; protein ubiquitination; cytokine; synovial fibroblasts; inflammation; TNF signal transduction
*
Address correspondence to: Salahuddin Ahmed, Ph.D., Department of Pharmaceutical Sciences, Washington State University College of Pharmacy, SPBS Room 411, 412 E. Spokane Falls Blvd, Spokane, WA-99210, Tel: (509)368-6566, [email protected]. Authors’ contributions N.A., A.K.S., and S. A. designed the research and wrote the paper; N.A. and A.K.S. performed the research. N.A., A.K.S., and S.A. analyzed and interpreted the data; S.A. provided his expertise, funding support, and gave critical suggestions while drafting of the manuscript.
Conflict of interest The authors declare no conflict of interest.
Akhtar et al.
Page 2
Author Manuscript Author Manuscript
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).
Author Manuscript
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.
Author Manuscript
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 J Immunol. Author manuscript; available in PMC 2017 September 15.
Akhtar et al.
Page 3
Author Manuscript
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 downregulating TNF-α signaling by influencing the protein ubiquitination pathway in RASFs.
Author Manuscript
Materials and Methods Reagents and Antibodies
Author Manuscript
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 anticIAP2 (#3031), rabbit anti-USP2 (#8036), rabbit monoclonal anti-RAD23A (#24555), antiK63 polyubiquitin (#5621), and anti-K48 polyubiquitin (#8081), anti-STAT-3 (#9132), antiNF-κBp65 (#8242), anti-p-c-Jun (S73) (#9164), anti-p-p38 (T180/Y182) (#4511), anti-pJNK (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
Author Manuscript
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).
J Immunol. Author manuscript; available in PMC 2017 September 15.
Akhtar et al.
Page 4
Author Manuscript
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
Author Manuscript
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
Author Manuscript
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 12myristate 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
Author Manuscript
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 ΔΔCt method.
J Immunol. Author manuscript; available in PMC 2017 September 15.
Akhtar et al.
Page 5
RNA isolation, reverse transcription, and miR-17 qRT-PCR in serum samples
Author Manuscript Author Manuscript
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
Author Manuscript
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
J Immunol. Author manuscript; available in PMC 2017 September 15. Published in final edited form as: J Immunol. 2016 September 15; 197(6): 2219–2228. doi:10.4049/jimmunol.1600360.
MicroRNA-17 Suppresses TNF-α Signaling by Interfering with TRAF2 and cIAP2 Association in Rheumatoid Arthritis Synovial Fibroblasts Nahid Akhtar#, Anil Kumar Singh#, and Salahuddin Ahmed#,* #Department
Author Manuscript
of Pharmaceutical Sciences, Washington State University College of Pharmacy, Spokane, 99204, Washington, United States
Abstract
Author Manuscript
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.
Keywords
Author Manuscript
Rheumatoid Arthritis; microRNA; protein ubiquitination; cytokine; synovial fibroblasts; inflammation; TNF signal transduction
*
Address correspondence to: Salahuddin Ahmed, Ph.D., Department of Pharmaceutical Sciences, Washington State University College of Pharmacy, SPBS Room 411, 412 E. Spokane Falls Blvd, Spokane, WA-99210, Tel: (509)368-6566, [email protected]. Authors’ contributions N.A., A.K.S., and S. A. designed the research and wrote the paper; N.A. and A.K.S. performed the research. N.A., A.K.S., and S.A. analyzed and interpreted the data; S.A. provided his expertise, funding support, and gave critical suggestions while drafting of the manuscript.
Conflict of interest The authors declare no conflict of interest.
Akhtar et al.
Page 2
Author Manuscript Author Manuscript
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).
Author Manuscript
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.
Author Manuscript
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 J Immunol. Author manuscript; available in PMC 2017 September 15.
Akhtar et al.
Page 3
Author Manuscript
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 downregulating TNF-α signaling by influencing the protein ubiquitination pathway in RASFs.
Author Manuscript
Materials and Methods Reagents and Antibodies
Author Manuscript
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 anticIAP2 (#3031), rabbit anti-USP2 (#8036), rabbit monoclonal anti-RAD23A (#24555), antiK63 polyubiquitin (#5621), and anti-K48 polyubiquitin (#8081), anti-STAT-3 (#9132), antiNF-κBp65 (#8242), anti-p-c-Jun (S73) (#9164), anti-p-p38 (T180/Y182) (#4511), anti-pJNK (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
Author Manuscript
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).
J Immunol. Author manuscript; available in PMC 2017 September 15.
Akhtar et al.
Page 4
Author Manuscript
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
Author Manuscript
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
Author Manuscript
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 12myristate 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
Author Manuscript
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 ΔΔCt method.
J Immunol. Author manuscript; available in PMC 2017 September 15.
Akhtar et al.
Page 5
RNA isolation, reverse transcription, and miR-17 qRT-PCR in serum samples
Author Manuscript Author Manuscript
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
Author Manuscript
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
