Supplementary data:

A microRNA signature associated with chondrogenic lineage commitment

Behnaz Bakhshandeh, Masoud Soleimani, Seyed Hassan Paylakhi and Nasser Ghaemi

J. Genet. 91, 171–182

Cell culture of 5 nM or a final miRNA inhibitor concentration of 50 nM after addition of cells to complexes in step 4). Briefly, 5 mL of cord blood, with informed consent of 2. Then we added 0.75 μL of transfection reagent to the mothers, was laid over 1.077 g/mL Ficoll-Hypaque 24.25 μL of culture medium without serum. In contin- (Pharmacia Biotech, Piscataway, USA) gradient and cen- uation, we included the diluted transfection reagent to trifuged at 400 g for 25 min. Mononuclear cells were then the prespotted miRNA mimic/inhibitor. separated and cultured in low glucose Dulbecco’s modi- 3. Incubation for 5–10 min at room temperature (15– fied Eagle’s medium (DMEM, GIBCO-BRL, Grand Island, 25◦C) to allow formation of transfection complexes USA) supplemented with 30% foetal bovine serum (FBS, was done. Gibco), dexamethasone (100 nM; Sigma), L-glutamine 4. After that 2 × 104 cells in 175 μLofgrowthmedium (2 mM; Gibco), penicillin (100 U/mL; Gibco), and strep- (containing serum and antibiotics) were seeded into the tomycin (0.1 mg/mL; Gibco) under humidified atmosphere ◦ well, on top of the miRNA mimic/inhibitor – reagent of 95% air with 5% CO2 at 37 C. The colonies of USSCs transfection complexes and incubated under mild shak- appeared after almost two weeks. When cells reached 80% ing for 6 h. Finally we changed the medium with fresh confluency, they were detached with 0.25% trypsin-EDTA growth medium till 72 h. (Gibco) and replated 1:3. Cells were diluted to 1 million cells per vial and frozen in FBS (Gibco) plus 10% dimethyl sulphoxide at a controlled rate to 70◦C by using a cell freezer PCR details box. Cell stocks were transferred to liquid nitrogen stor- ◦ age after 24 h. In chondrogenic differentiation process, tube Standard procedure for PCR: denaturation at 94 C for 15 s, annealing at 53–61◦C for 30 s according to the primers, and caps were loose for air circulation. Half of the differentiation ◦ medium was changed every 2–3 days for 21 days. extension at 72 C for 45 s. The number of cycles varied between 30 and 40, depending on the abundance of partic- ular mRNA. Reaction mixtures for PCR included 2.5 μL cDNA, 1× PCR buffer (AMS TM, Cinnagen, Iran), 200 μM μ miRNA transfection procedure dNTPs, 0.5 M of each of forward and reverse primers and 1U Taq DNA polymerase (Fermentas, USA). Amplified 1. We spotted 0.5 μL miRNA mimic or inhibitor in 2 μL DNA fragments were electrophoresed on 2% agarose gel. of RNAase-free water into a single well of a 96-well The gels were stained with ethidium bromide (10 μg/mL) plate (this will give a final miRNA mimic concentration and photographed on a UV trans-illuminator (uvidoc).

Journal of Genetics, Vol. 91, No. 2, August 2012 Supplementary data, J. Genet. 91, 171–182

Primers used for RT-PCR and miRNA qPCR.

Primer sequences ◦ Gene Sense, top; antisense, bottom Tm ( C)

HPRT CCT GGC GTC GTG ATT AGT G 58 TCA GTC CTG TCC ATA ATT AGT CC ACAN AAG ACG GCT TCC ACC AGT G 54.6 AAA GAC CTC ACC CTC CAT CTC COL2A1 GCC TGG TGA TGA TGG TGA AG 54.8 GCC TGG ATA ACC TCT GTG AC SOX9 GCA GGA GGA GAA GAG AAG G 52 GAG GCG AAT TGG AGA GGA G BMP6 CTC GGG GTT CAT AAG GTG AA 53.7 ACA GCA TAA CAT GGG GCT TC TFGbR GCA ATG GGC TTA GTA TTC TG 58 CAT TAC TCT CAA GGC TTC ACA G NLK CCT CAT AAA CAG CCA TCT CTT C 59 ATA TCG TAG TCG CCC TTC ATC mir-1268 CGGGCGTGGTGGT 55.7 mir-630 AGTATTCTGTACCAGGGAAGGT 54.9 mir-376a ATCATAGAGGAAAATCCACGT 51.2 mir-624 GCACAAGGTATTGGTATTACCT 52.4

Fold change calculation DIANA-mirPath web-based computational tool performs an enrichment analysis of multiple miRNA target genes For additional information about the chip, please see: (predicted by different algorithms, i.e. DIANA-microT 3.0, http://www.chem.agilent.com/en-US/Store/_layouts/Agilent/ PicTar, TargetScan5) comparing each set of miRNA tar- Commerce/ProductDetail.aspx?productID=G4470B&cat= gets to all known KEGG pathways (http://diana.cslab. miRNA&pcat=DNAMicroarraysSub&rcat=DNAMicroarrays. ece.ntua.gr/). Briefly, 500 ng of total RNA from each sample were To acquire specific pathways particularly controlled by chemically labelled by dephosphorylating using calf intesti- highest altered miRNAs, in silico analysis between the eight nal alkaline phosphatase (CIP) and ligating cyanine3-pCp by top modulated miRNAs (mir-143, mir-145, mir-188–5p, mir- a T4-RNA ligase using Agilent miRNA Complete Labelling 221, mir-376a, mir-376c, mir-630 and mir-377) targets and and Hyb kit (p/n5190–0456). Labelled samples were dried the most common signalling pathways by using DIANAmir- μ and resuspended in 18 L of nuclease-free water and Path was performed. cohybridized with in situ hybridization buffer (Agilent) for Figure 2 shows the number of genes targeted by each 20 h at 55◦C and washed at room temperature for 5 min in ◦ miRNA, the ‘union’ bar indicates the sum of all genes Gene Expression Wash Buffer 1 (Agilent) and 5 min at 37 C belonging to a specific pathway predicted to be targeted by in Gene Expression Wash Buffer 2 (Agilent). the eight considered miRNAs. As revealed, genes putatively We calculated the fold change in the following way: interacting with these miRNAs were found to be mostly FC = sign (LR) × 2∧ |LR| , involved in some specific signalling pathways including TGF-beta, Wnt, MAPK, ErbB, mTOR, p53, GnRH, Notch, where LR, log2ratio = log2 (condition2/condition1): condi- Insulin, and other pathways such as adipocytokine, melano- tion1 or 2 corresponding to the mean of the replicates for genesis, axon guidance, dorsa-ventral axis formation, cell their respective conditions; sign (LR), sign of the log2ratio; adhesion molecules, adherens junction, tight junction, focal ∗, multiplies; ∧, exponent (power); |LR|, absolute value of adhesion, regulation of actin cytoskeleton, ubiquitin medi- the log2ratio. ated proteolysis and cell cycle.

Journal of Genetics, Vol. 91, No. 2, August 2012 Supplementary data, J. Genet. 91, 171–182

Figure 1.

Journal of Genetics, Vol. 91, No. 2, August 2012 Supplementary data, J. Genet. 91, 171–182

Figure 2.

Gene ontology The last in silico analysis to illuminate the molecular and cellular mechanisms of chondrogenesis was gene ontology (GO). Based on our findings, 32 GOs were classified on the basis of targets of the 60 top modulated miRNAs. Shown in this figure, more than half of target genes of these miRNAs were involved in protein binding, transferase activity, metal ion binding, hydrolase activity and nucleotide binding. All cellular mechanisms in GO annotation are divided into three major groups: cellular component, molecular function and biological process. Most of the annotated GOs involved in chondrogenesis belonged to ‘binding or activity’ subgroups Figure 3. Gene ontology based on chondrogenesis related microR- of ‘molecular function’ group. This finding is in congruence NAs. with the nature of differentiation process, which is mostly the consequence of alterations in molecular functions of the cell.

Journal of Genetics, Vol. 91, No. 2, August 2012 Supplementary data, J. Genet. 91, 171–182

Table 1 . List of 60 miRNAs and their FCs are in the selected area (FC>3orFC<−3).

Standard miRNA Average FC deviation hsa-miR-100 −3.02696 0.037265 hsa-miR-103 −3.35768 – hsa-miR-10b −3.07584 0.222548 hsa-miR-1185 −3.59495 – hsa-miR-1207-5p 4.128908 0.405995 hsa-miR-1225-3p 3.561872 0.217463 hsa-miR-1225-5p 4.805859 0.22927 hsa-miR-1228 3.258105 – hsa-miR-1246 4.441199 0.40673 hsa-miR-1265 50.35625 – hsa-miR-1268 20.84915 0.276272 hsa-miR-127-3p −3.00775 0.31791 hsa-miR-1274a −3.2 – hsa-miR-1275 3.46149 0.205122 hsa-miR-1287 3.795 0.291645 hsa-miR-1290 6.388892 0.286554 hsa-miR-1306 3.25 0.365301 hsa-miR-130a −3.44979 0.226885 hsa-miR-139-3p −7.81545 – hsa-miR-143 −4.05566 0.435916 hsa-miR-145 −3.74297 0.311982 hsa-miR-146a 3.49434 0.158473 hsa-miR-149 3.545871 – hsa-miR-152 −3.45 – hsa-miR-155 −3.00046 – hsa-miR-16 −3.01525 0.131476 hsa-miR-181a 3.371605 0.101744 hsa-miR-181b 3.050069 – hsa-miR-181d 3.4 0.329126 hsa-miR-188-5p 8.595992 0.419512 hsa-miR-1915 7.303356 0.427945 hsa-miR-210 3.579726 0.243003 hsa-miR-221 −4.51474 0.378457 hsa-miR-29b −3.10593 0.053065 hsa-miR-337-5p −3.0689 – hsa-miR-365 −3.39544 – hsa-miR-374a −3.72961 – hsa-miR-376a −4.89135 0.639909 hsa-miR-376a∗ −3.00547 – hsa-miR-376c −4.57688 0.63815 hsa-miR-377 −4.50125 0.672487 hsa-miR-379 −3.77914 0.237569 hsa-miR-490-5p −3.08144 0.137099 hsa-miR-515-5p 3.821597 – hsa-miR-516a-5p −3.2494 – hsa-miR-570 8.857679 – hsa-miR-595 3.086211 0.204763 hsa-miR-613 7.568021 – hsa-miR-623 3.5 0.335718 hsa-miR-624 76.52025 – hsa-miR-630 15.76529 1.356392 hsa-miR-637 3.361374 – hsa-miR-638 3.233647 0.140096 hsa-miR-654-3p −3.45632 – hsa-miR-744 −5.99377 – hsa-miR-887 11.78641 – hsa-miR-940 3.004555 – hsa-miR-99b∗ 3.171858 –

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Table 2 . Complete information about putative targets.

Gene Putative target genes Pathway hsa-let7f FZD4 frizzled homologue 4 WNT signalling GALC galactosylceramidase GAG synthesis B3GNT1 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1 GAG synthesis ACTR10 actin-related protein 10 homolog Actin cytoskeleton FARP1 FERM, RhoGEF (ARHGEF) and pleckstrin domain protein 1 WNT signalling (chondrocyte-derived) PDGFB platelet-derived growth factor beta polypeptide MAPK signalling CALM1 calmodulin 1 (phosphorylase kinase, delta) Adherens Junction TGFBR1 transforming growth factor, beta receptor 1 TGF-beta signalling hsa-miR-130a BMPR2 bone morphogenetic protein receptor, type II TGF-beta signalling SMAD5 SMAD family member 5 TGF-beta signalling BMP3 bone morphogenetic protein 3 TGF-beta signalling TGFBR1 transforming growth factor, beta receptor I TGF-beta signalling WNT10A wingless-type MMTV integration site family, member 10A WNT signalling PDGFRA platelet-derived growth factor receptor, alpha polypeptide MAPK signalling SMAD4 SMAD family member 4 TGF-beta signalling SOX5 SRY-box 5 Cartilage formation PPARG peroxisome proliferator-activated receptor gamma PPAR signalling hsa-mir-143 MAP3K7 mitogen-activated protein kinase kinase kinase 7 MAPK signalling CHST10 carbohydrate sulfotransferase 10 Sulfation of chondroitin MAPK7 mitogen-activated protein kinase 7 MAPK signalling COL1A1 collagen, type I, alpha 1 Cartilage formation MAPK1 mitogen-activated protein kinase 1 MAPK signalling COL5A1 collagen, type V, alpha 1 Cartilage formation PDGFRA platelet-derived growth factor receptor, alpha polypeptide MAPK signalling SMAD3 SMAD family member 3 TGF-beta signalling CTNND1 catenin (cadherin-associated protein), delta 1 Adherens Junction COL5A2 collagen, type V, alpha 2 Cartilage formation ERBB3 v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 ERBB signalling hsa-mir-145 SOX9 SRY-box 9 Cartilage formation SOX11 SRY-box 11 Cartilage formation SMAD3 SMAD family member 3 TGF-beta signalling SMAD5 SMAD family member 5 TGF-beta signalling TGFBR2 transforming growth factor, beta receptor II TGF-beta signalling SMAD4 SMAD family member 4 TGF-beta signalling MAP4K4 mitogen-activated protein kinase kinase kinase kinase 4 MAPK signalling MAP2K4 mitogen-activated protein kinase kinase 4 MAPK signalling PDGFD platelet derived growth factor D MAPK signalling CDH2 cadherin 2, type 1 Adherens Junction CTNND1 catenin (cadherin-associated protein), delta 1 Adherens Junction P4HA1 procollagen-proline, 2-oxoglutarate 4-dioxygenase alpha Cartilage formation MAP4K2 mitogen-activated protein kinase kinase kinase kinase 2 MAPK signalling MAP3K3 mitogen-activated protein kinase kinase kinase 3 MAPK signalling MAP3K11 mitogen-activated protein kinase kinase kinase 11 MAPK signalling ERBB4 v-erb-a erythroblastic leukemia viral oncogene homolog 4 ERBB signalling hsa-miR-181a TGFBRAP1 transforming growth factor, beta receptor associated protein 1 TGF-beta signalling TGFBR1 transforming growth factor, beta receptor I TGF-beta signalling TNFRSF11B osteoprotegerin MAPK signalling PCDHAC1 protocadherin alpha subfamily C, 1 Adherens Junction PCDHAC2 protocadherin alpha subfamily C, 2 Adherens Junction PCDHA1 protocadherin alpha 1 Adherens Junction PCDHA10 protocadherin alpha 10 Adherens Junction PCDHA11 protocadherin alpha 11 Adherens Junction PCDHA12 protocadherin alpha 12 Adherens Junction PCDHA13 protocadherin alpha 13 Adherens Junction PCDHA2 protocadherin alpha 2 Adherens Junction PCDHA3 protocadherin alpha 3 Adherens Junction PCDHA4 protocadherin alpha 4 Adherens Junction PCDHA5 protocadherin alpha 5 Adherens Junction PCDHA6 protocadherin alpha 6 Adherens Junction

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Table 2 (contd).. (contd.).

Gene Putative target genes Pathway

PCDHA7 protocadherin alpha 7 Adherens Junction PCDHA8 protocadherin alpha 8 Adherens Junction PDGFRA platelet-derived growth factor receptor, alpha polypeptide MAPK signalling BMP3 bone morphogenetic protein 3 TGF-beta signalling SOX5 SRY-box 5 Cartilage formation MAP3K3 mitogen-activated protein kinase kinase kinase 3 MAPK signalling MAP3K7IP3 MAP kinase kinase kinase 7 interacting protein 3 MAPK signalling PDAP1 PDGFA associated protein 1 MAPK signalling NOTCH2 Notch homolog 2 Notch signalling MAPK1IP1L mitogen-activated protein kinase 1 interacting protein 1-like MAPK signalling BMPR2 bone morphogenetic protein receptor, type II TGF-beta signalling SMAD2 SMAD family member 2 TGF-beta signalling MADD MAP-kinase activating death domain MAPK signalling CDH13 cadherin 13 Adherens Junction ACAN aggrecan Cartilage Formation MAP3K10 mitogen-activated protein kinase kinase kinase 10 MAPK signalling PCDHB6 protocadherin beta 6 Adherens Junction MAP4K4 mitogen-activated protein kinase kinase kinase kinase 4 MAPK signalling hsa-mir-188–5p PCDH9 protocadherin 9 Adherens Junction MAP3K7IP3 MAP kinase kinase kinase 7 interacting protein 3 MAPK signalling MAP3K3 mitogen-activated protein kinase kinase kinase 3 MAPK signalling MAP2K4 mitogen-activated protein kinase kinase 4 MAPK signalling SMAD2 SMAD family member 2 TGF-beta signalling P4HB procollagen-proline, 2-oxoglutarate 4-dioxygenase beta Cartilage Formation MAP3K9 mitogen-activated protein kinase kinase kinase 9 MAPK signalling hsa-mir-376a BMPR2 bone morphogenetic protein receptor, type II TGF-beta signalling TGFBR1 transforming growth factor, beta receptor I TGF-beta signalling PCDH17 protocadherin 17 MAPK signalling hsa-mir-376c SOX11 SRY (sex determining region Y)-box 11 Cartilage formation CDH11 cadherin 11, type 2 Adherens Junction MAP3K7IP2 mitogen-activated protein kinase kinase kinase 7 interacting protein 2 MAPK signalling SOX4 SRY-box 4 Cartilage formation MADD MAP-kinase activating death domain MAPK signalling PCOLCE2 procollagen C-endopeptidase enhancer 2 Cartilage Formation PCDH11X protocadherin 11 X-linked Adherens Junction PCDH11Y protocadherin 11 Y-linked Adherens Junction MAP3K7 mitogen-activated protein kinase kinase kinase 7 MAPK signalling PCDH10 protocadherin 10 Adherens Junction SOX11 SRY-box 11 Cartilage formation hsa-mir-624 SMAD2 SMAD family member 2 TGF-beta signalling WNT5A wingless-type MMTV integration site family, member 5A WNT signalling PCDH19 protocadherin 19 Adherens Junction CTNNB1 catenin (cadherin-associated protein), beta 1 Adherens Junction hsa-mir-630 TOB2 transducer of ERBB2, 2 ERBB signalling GJC1 gap junction protein, gamma 1, 45kDa Gap junction PDGFRA platelet-derived growth factor receptor, alpha polypeptide MAPK signalling TGFBR2 transforming growth factor, beta receptor II TGF-beta signalling hsa-mir-1268 SOX12 SRY-box 12 Cartilage formation CAMK2G calcium/calmodulin-dependent protein kinase II Adherens Junction hsa-miR-29b COL3A1 collagen, type III, alpha 1 Cartilage formation COL4A5 collagen, type IV, alpha 5 Cartilage formation COL4A1 collagen, type IV, alpha 1 Cartilage formation COL7A1 collagen, type VII, alpha 1 Cartilage formation COL15A1 collagen, type XV, alpha 1 Cartilage formation COL2A1 collagen, type II, alpha 1 Cartilage formation COL4A6 collagen, type IV, alpha 6 Cartilage formation CSGALNACT2 chondroitin sulfate N-acetylgalactosaminyltransferase 2 Cartilage formation SOX12 SRY-box 12 Cartilage formation MAP2K6 mitogen-activated protein kinase kinase 6 MAPK signalling

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Table 2 (contd).. (contd.).

Gene Putative target genes Pathway

TGIF2 TGFB-induced factor homeobox 2 TGF-beta signalling SERPINH1 serpin peptidase inhibitor (collagen binding protein 1) Cartilage Formation NOTCH2 Notch homolog 2 Notch signalling PPARD peroxisome proliferator-activated receptor delta PPAR signalling hsa-mir-221 CHSY1 chondroitin sulfate synthase 1 Sulfation of chondroitin CAMKK1 calcium/calmodulin-dependent protein kinase kinase 1, alpha Adherens Junction ERBB4 v-erb-a erythroblastic leukemia viral oncogene homolog 4 ERBB signalling SOX11 SRY -box 11 Cartilage formation ACVR2B activin A receptor, type IIB TGF-beta signalling MAPK10 mitogen-activated protein kinase 10 MAPK signalling PCDHAC1 protocadherin alpha subfamily C, 1 Adherens Junction PCDHAC2 protocadherin alpha subfamily C, 2 Adherens Junction PCDHA1 protocadherin alpha 1 Adherens Junction PCDHA10 protocadherin alpha 10 Adherens Junction PCDHA11 protocadherin alpha 11 Adherens Junction PCDHA12 protocadherin alpha 12 Adherens Junction PCDHA13 protocadherin alpha 13 Adherens Junction PCDHA2 protocadherin alpha 2 Adherens Junction PCDHA3 protocadherin alpha 3 Adherens Junction PCDHA4 protocadherin alpha 4 Adherens Junction PCDHA5 protocadherin alpha 5 Adherens Junction PCDHA6 protocadherin alpha 6 Adherens Junction PCDHA7 protocadherin alpha 7 Adherens Junction PCDHA8 protocadherin alpha 8 Adherens Junction PCDHAC1 protocadherin alpha subfamily C, 1 Adherens Junction PCDHAC2 protocadherin alpha subfamily C, 2 Adherens Junction PCDHA1 protocadherin alpha 1 Adherens Junction PCDHA10 protocadherin alpha 10 Adherens Junction PCDHA11 protocadherin alpha 11 Adherens Junction PCDHA12 protocadherin alpha 12 Adherens Junction PCDHA13 protocadherin alpha 13 Adherens Junction PCDHA2 protocadherin alpha 2 Adherens Junction PCDHA3 protocadherin alpha 3 Adherens Junction PCDHA4 protocadherin alpha 4 Adherens Junction PCDHA5 protocadherin alpha 5 Adherens Junction PCDHA6 protocadherin alpha 6 Adherens Junction PCDHA7 protocadherin alpha 7 Adherens Junction PCDHA8 protocadherin alpha 8 Adherens Junction PDGFA platelet-derived growth factor alpha polypeptide MAPK signalling

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