Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most
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1 Supplementary Figure 1 MicroRNA expression in human synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most differentially expressed between human synovial fibroblasts (SF) from different joint locations were measured by qpcr in an independent set of rheumatoid arthritis (RA) and osteoarthritis (OA) SF isolated from knee, shoulder or hand joints. Open shapes are OA SF, closed shapes are RA SF. *=p<0.05; **=p<0.01; ***=p<0.001;****=p< by two-way ANOVA with Bonferroni correction.
2 H O T A IR HOTAIR 9 C t H O T A IR -B 2 M H ip K n e e T o e Supplementary Figure 2 The expression of HOTAIR in synovial fibroblasts from lower extremity joints. Human synovial fibroblasts (SF) from distal joint locations express more HOTAIR than SF from proximal joint locations (knee, hip). The expression of HOTAIR was measured by qpcr with normalization to beta2 microglobulin (B2M) in SF obtained from hip, knee and toe (proximal interphalangeal joints from digits II-IV) joints of patients with rheumatoid arthritis and osteoarthritis. Due to a limited access, a toe sample could be obtained from only one patient.
3 Supplementary Figure 3 MicroRNA expression in mouse synovial fibroblasts from different locations. MicroRNA, which were identified by RNAseq as most differentially expressed between human synovial fibroblasts (SF) from different joint locations were measured by qpcr in SF isolated from joints of wildtype (wt) and TNF transgenic (tg) C57BL/6 mice. ****=p< by two-way ANOVA with Bonferroni correction.
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10 Supplementary Figure 4 Location specific histone marks in HOXA and HOXC clusters in synovial fibroblasts (SF). Regulation of the positional HOX signatures in knee (blue), shoulder (grey) and hand (orange) SF by histone modifications. H3K27me3, H3K4me3 and H3K27ac marks were measured by chromatin immunoprecipitation DNA sequencing (ChIPseq) in SF from knee, shoulder and hand joints of osteoarthritis (OA) and rheumatoid arthritis (RA) patients (n=1 each) and from knee of healthy (H) subject (n=1) with joint pain. Coverage values of H3K27me3, H3K4me3 and H3K27ac in HOXA and HOXC as measured by ChIP-Seq. Profiles of input samples are shown below each sample (light color). Data is visualized using Integrative Genomics Viewer (IGV).
11 a Development_Ossification and bone remodeling b Proteolysis_Connective tissue degradation Supplementary Figure 5 MetaCore pathway analysis of transcriptome data from synovial fibroblasts (SF) from knee, shoulder and hand. (a-b) Process Network enrichment analysis of RNA sequencing data (n=21) between joints of upper and lower extremities and between specific joint locations (knee vs hand, knee vs shoulder, hand vs shoulder) using MetaCore from Thomson Reuters and default settings (threshold of log (fold change) in each comparison set to 0.5, p=0.05, FDR<0.05. Heatmaps of the enriched genes for selected arthritis-relevant process networks present among significantly enriched process networks between locations (a) Joint-specific network: Development_Ossification and bone remodeling and (b) Arthritis-relevant network: Proteolysis_Connective tissue degradation».
12 x -fo ld G a p m e r H O T A IR 1 0 * *** 0 H O T A IR M M P 1 Supplementary Figure 6 Expression of matrix metalloproteinase 1 (MMP1) in HOTAIR-silenced knee synovial fibroblasts. Silencing of HOTAIR in knee OA SF using Antisense LNA TM HOTAIR GapmeR (48 h) resulted in increased expression of matrix metalloproteinase 1 (MMP1) (n=9, qpcr, data presented as box and whiskers: min to max). X-fold change of Antisense LNA TM HOTAIR GapmeR transfected cells over Antisense LNA TM GapmeR Negative Control A transfected cells (set to 1 as depicted by a dotted line). Statistics: two tailed paired t- test, * p<0.05; ***p<0.001.
13 a b M M P 1 M M P C t t a r g e t - H P R T C t t a r g e t - H P R T H a n d S h o u ld e r K n e e H a n d S h o u ld e r K n e e Supplementary Figure 7 Expression of matrix metalloproteinase 1 (MMP1) and MMP3 in synovial tissues from different joints. The expression of (a) matrix metalloproteinase 1 (MMP1) and (b) MMP3 did not differ between synovial tissues from hand, shoulder and knee joints of RA and OA patients, qpcr. Statistics: 1way ANOVA with Bonferroni s post test. Open shapes are OA SF, closed shapes are RA SF.
14 Supplementary Table 1 Characteristics of patients from which synovial fibroblasts were obtained for positional RNA sequencing (n=21) Treatment Joint Diagnosis Passage Sex Age (years) Disease duration (years) knee OA 5 M 64 NA knee OA 5 F 61 NA knee OA 7 F 72 NA knee RA 6 F DMARD, biologic, NSAID, corticosteroid knee RA 5 F 64 7 DMARD, biologic knee RA 6 F DMARD, NSAID, corticosteroid knee arthralgia 7 F 59 knee arthralgia 7 F 49 knee arthralgia 7 M 42 shoulder OA 6 F 88 NA shoulder OA 7 F 78 NA shoulder OA 7 F 68 NA shoulder RA 6 F Biologic, corticosteroid shoulder RA 6 F Biologic shoulder RA 6 F DMARD MCP OA 6 M 68 NA CMC OA 6 F 61 NA CMC OA 6 F 55 NA MCP RA 6 F DMARD MCP RA 5 F DMARD, NSAID MCP RA 6 F DMARD, corticosteroid OA = osteoarthritis, RA = rheumatoid arthritis, F = female, M= male, NA = not assessed; DMARD = disease modifying antirheumatic drug; NSAID = non-steroidal anti-inflammatory drug; MCP = metacarpophalangeal joint; CMC = carpometacarpal joint.
15 Supplementary Table 2 Patient s characteristics for synovial fibroblasts used for measurements of HOX gene expression by qpcr Joint Diagnosis N Sex Age (years; Disease duration Treatment min-max) (years; min-max) (number of patient receiving drug) hands (MCP, wrist) RA 4 F 69 (51-77) 22 (15-50) NSAID (1), DMARD (4), corticosteroid (1) hands (CMC) OA 4 3F/1M 61 (53-74) NA elbow RA 7 F 56 (40-80) 18 (8-27) NSAID (4), DMARD (2), corticosteroids (2), biologics (3) elbow OA 1 F 52 NA shoulder RA 4 3F/1M 72 (68-78) 32 (17-38) DMARD (1), corticosteroids (2), biologics (3) shoulder OA 4 3F/1M 67 (64-95) NA hip RA 3 2F/1M 69 (67-73) 12 (2-28) NSAID (1), DMARD (3), corticosteroids (1) hip OA 3 1F/2M 60 (58-68) NA knee RA 4 2F/2M 72 (61-79) 20 (5-51) NSAID (1), DMARD (2), corticosteroids (3), biologics (1) knee OA 4 2F/2M 66 (61-77) NA knee arthralgia 3 2F/1M 49 (42-59) ankle RA 5 3F/2M 55 (27-81) <1 DMARD (1) ankle resolving arthritis 2 F 52 (52-52) NA N = number of patients from which synovial fibroblast were obtained per each location; RA = rheumatoid arthritis, OA = osteoarthritis, NA = not assessed; DMARD = disease modifying anti-rheumatic drug; NSAID = non-steroidal anti-inflammatory drug; MCP = metacarpophalangeal joint; CMC = carpometacarpal joint.
16 Supplementary Table 3 Patient s characteristics for synovial tissues used for qpcr measurements of HOX transcripts Joint Diagnosis N Sex Age (years; min-max) Disease duration (years; min-max) Treatment hands (MCP, wrist) RA 5 F 62 (52-69) 21 (8-50) NSAID (2), DMARD (2), corticosteroids (1) elbow RA 6 4F/2M 54 (50-65) 16 (13-25) NSAID (2), DMARD (3), corticosteroids (1), biologic (1) shoulder RA 4 3F/1M 69 (65-70) 24 (4-48) NSAID (2), DMARD (1), corticosteroid (1), biologic (1) shoulder OA 4 3F/1M 67 (64-75) NA hip RA 2 F 77 (74-79) 42 (44-44) NSAID (2) hip OA 4 1F/2M 63 (50-69) NA knee RA 4 2F/2M 63 (40-71) 20 (9-51) NSAID (1), DMARD (3), corticosteroids (3), biologics (2) knee OA 4 2F/2M 67 (64-72) NA NSAID (2) ankle JRA 1 F NSAID (1), DMARD (1) ankle RA 2 F 53 (39-66) 14 (9-18) NSAID (1), DMARD (1), corticosteroid (1) N=number of tissues per location, RA = rheumatoid arthritis, OA = osteoarthritis; NA= not assessed; F= female, M = male, DMARD = disease modifying anti-rheumatic drug; NSAID = non-steroidal anti-inflammatory drug; MCP = metacarpophalangeal joint
17 Supplementary Table 4 Characteristics of healthy wild type and arthritic TNF transgenic mice used for isolation of synovial fibroblasts Sex Age (weeks) when sacrificed Joint of origin of synovial fibroblasts Front paw Knee Ankle Wt C57BL/6 1 F F F F F F F M M M TghuTNF C57BL/6 1 F M F F F F F F M M 8 + Wt = wild type, TghuTNF = TNF transgenic, F = female, M = male
18 Supplementary Table 5 Patient s characteristics for rheumatoid arthritis synovial tissues used for scoring by Krenn synovitis score Joint Diagnosis N Sex Age Mean years (min-max) Disease duration Mean years (min-max) Treatment (number of patients receiving treatment) hands RA 15 14F/1M 51 (33-64) 16 (5-36) NSAID (9), DMARD (11), corticosteroid (6), biologics (2), untreated (2) (MCP, PIP) JRA 2 F 40 (38-42) 23 (1 NA) NSAID (2), DMARD (2), corticosteroid (1), biologics elbow RA 6 5F/1M 65 (50-75) 38 (28-49) NSAID (4), DMARD (2), corticosteroids (2), untreated (1) shoulder RA 5 2F/3M 64 (51-71) 54 (30-66) NSAID (3), DMARD (3), corticosteroid (3), biologics (2) JIA 2 2M 44 (31-58) 9 (6-11) NSAID (1), DMARD (2), corticosteroid (1), biologics (1) hip RA 4 3F/1M 71 (67-78) 43 (38-55) NSAID (2), DMARD (3), corticosteroids (3) JIA 2 M 33 (32-33) 8 (2-13) corticosteroids (2) knee RA 6 3F/3M 68 (60-76) 45 (30-66) (1 NA) NSAID (3), DMARD (3), corticosteroids (3) JIA 1 M NSAID (1), DMARD (1), corticosteroids (1) N= number of synovial tissues per location; F = female, M = male; NA = not assessed; DMARD = disease modifying anti-rheumatic drug; NSAID = non-steroidal anti-inflammatory drug; MCP = metacarpophalangeal joint; PIP = proximal interphalangeal joint; JIA = juvenile idiopathic arthritis, RA = rheumatoid arthritis, OA = osteoarthritis.
19 Supplementary Table 6 Sequences of human primers and probes for SybrGreen and TaqMan qpcr measurements HPRT1 For: 5 ATG GAC AGG ACT GAA CGT CTT G 3 Rev: 5 GGC TAC AAT GTG ATG GCC TC 3 B2M For : 5 TAG TTA AGT GGG ATC GAG ACA TG 3 Rev: 5 TGG AAT TCA TCC AAT CCA AAT GC 3 MMP1 For: 5 GCT AAC AAA TAC TGG AGG TAT GAT G 3 Rev:5 ATT TTG GGA TAA CCT GGA TCC ATA G 3 HOXA9 For: 5 ATC GAT CCC AAT AAC CCA GCA 3 Rev: 5 GAG TGG AGC GCG CAT GAA 3 HOXA10 For: 5 CCC TTC CGA GAG CAG CAA A 3 Rev: 5 GAA TTG CCC AGG GAA TCC TTC T 3 HOXA11 For: 5 GTC TTC CGG CCA CAC TGA G 3 Rev: 5 TGC GTT GGC CAC TGG AG 3 HOXA11-AS For: 5 TTC TCC TGG GAG GTC CAA CAG 3 Rev: 5 AGA AAT GGA ACT CGG ACT TGG C 3 HOXA13 For: 5 CAG GCG CAG CCT CCC 3 Rev: 5 GAG ACC ACG TCG GGC AG 3 HOXC-AS1 For: 5 GAC CGA GCT TGA AGA AGT GTA GG 3 Rev: 5 AGG AAG ATG GGA TGG GGA CG 3 HOXC-AS3 For: 5 TGC GAG AGA CCA ATG GGA TTT G 3 Rev: 5 ACA ACT TGA CCC CGC TGA CG 3 HOXC4 For: 5 CCA GAG GGT TGG AAA TTA ATG ATC 3 Rev: 5 GGA CTG TGT TCA GGG ATG TAG C HOXC10 For: 5 TAT CTC CTC TTT CGC TTC GTT ATC C 3 Rev: 5 CAA TGA AAT CAA GAC GGA GCA GAG 3 HOXC8 For: 5 ACA CTC ACA ACT CTA ACT ACC TGT C 3 Rev: 5 GAG TCC GAA CTT TTC ACT GTT TCT C 3 HOXC-AS2 For: 5 CCA CCC AAG GAA ATT TGC AGT G 3 Rev: 5 TTA ATT GTA CTT CGG GCT CGT ATT G 3 HOXC11 For: 5 TAT CTG ACT CTC GCT GTG GGA C 3 Rev: 5 AGC AAG ACA TTG TCG CCG AGG 3 HOXC13 For: 5 AAG TCT CCC TTC CCA GAC GTG 3 Rev: 5 CTG CAC CTT AGT GTA GGG CAC 3 HOTTIP For: 5 CAC AAG CAG GTA CCA CAA AGC CA 3 Rev: 5 TCT CCT TGC TCA GGG ACT CT 3 AC For: 5 CTG GCA AAC TCT CCC AAC TTA G 3 Rev: 5 CTG ACT ACT CCG AAC TGC TTG 3 HOTAIR For: 5 GAT CCG AAA GCT TCC ACA GTG 3 Rev: 5 AAC TCC CAG GCC TCA GTG C 3 TNFa For: 5 TCC AGG CGG TGC TTG TTC 3 Rev: 5 GCC TGC CAC GAT CAG GAA 3 MMP13 For: 5` TCC TAC AAA TCT CGC GGG AAT 3 Probe: 5` CAT GGA GCT TGC TGC ATT CTC CTT CAG 3` Rev: 5`GCA TTT CTC GGA GCC TCT CA 3`
20 Supplementary Table 7 Sequences of mouse primers for SybrGreen qpcr measurements B2m For: 5 CTT CAG CAA GGA CTG GTC TTT C 3 Rev: 5 CAT GTC TCG ATC CCA GTA GAC G 3 Hoxa9 For: 5 CCA TCG ATC CCA ATA ACC CGG 3 Rev: 5 TCC TTC TCC AGT TCC AGC GTC 3 Hoxa11 For: 5 AGC GAG AGT TCT TCT TCA GCG 3 Rev: 5 ACT TGA CGG TCG GTG AGG TTG 3 Hoxa11os For: 5 AAT TTG GAG ACC CAC CTC AGG G 3 Rev: 5 CCA GCC AAC ATG AGT TAC ACC G 3 Hoxc8 For: 5 GCG AGC GTG GTG CAA TAT CC 3 Rev: 5 GAG CGT GGG GTC TCA TCC AT 3 Hoxc10 For: 5 ATC AAG ACA GAG CAA AGC CTG G 3 Rev: 5 GCC TTT ATC TCC TCT TTA GCT TCG 3 Hottip For: 5 GAG GTG CCT TCT CCC TTC TTT G 3 Rev: 5 GGA ATC AGA AGC AGG TAC AAC GG 3 Hotair For: 5 CGA CGC CTT CCT TAT AAG CTC 3 Rev: 5 CTC TCT CTG CCT TTA CAG TTG C 3
21 Supplementary Table 8 Control regions to assess specificity of immunoprecipitation reactions used for Chromatin Immunoprecipitation DNA sequencing Mark Positive region Negative region H3K4me3 GAPDH TSS, EIF4A2 promoter MB exon 2, Sat2 H3K27me3 MYT1, TSH2B GAPDH TSS, EIF4A2 promoter H3K4me1 ACTB3, GAS2L1 GAPDH TSS, EIF4A2 promoter H3K27ac ACTB promoter, EIF4A2 promoter MYT1, TSH2B
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