http / / cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology RT-PCR Runx d PCR RT-qPCR Western BMPs Wnt Ihh

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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 547 micrornas * / / microrna mrna β- RT-PCR Runx d micrornas mrnas PCR RT-qPCR Western 7 d 66 micrornas 640 mrna 94 micrornas 744 mrna 14 d 58 micrornas 781 mrna 96 micrornas 603 mrna PCR Western mir-29b TGFβ3 mir-29b TGFβ3 Runx2 microrna mrna BMPs Wnt Ihh microrna mrna Q291 MicroRNAs Involved in the Gene Regulation Related to the Osteogenic Differentiation of Ligament Fibroblasts ZHANG Xiu-Mei CUI Ya-Zhou ZHOU Xiao-Yan YU Fang-Cang HAN Jin-Xiang * Key Laboratory for Biotech-Drug of Health Ministry Key Laboratory for Medical Molecular Biology of Shandong Province Shandong Medical Biotechnological Center Shandong Academy of Medical Sciences Jinan China Abstract The osteogenic differentiations of ligament fibroblasts are closely associated with ectopic ossification-related diseases such as ankylosing spondylitis but the exact mechanism remained unclear To evaluate the microrna and mrna expression profiling of ligament fibroblasts during its osteogenic differentiation fibroblasts from ligments were isolated and cultured in osteogenic medium containing dexamethasone ascorbic acid and β-glycerol phosphate Two osteoblastic markers Osteocalcin and Runx2 were detected by RT-PCR at day 0 7 and 14 Microarray analyses were performed to profile the expression of micrornas and mrnas during the process of osteogenic differentiation Real-time quantitative PCR RT-qPCR and Western blotting were used to for further confirmation following bioinformatic analyses The results showed that 66 micrornas and 640 mrna were up-regulated * Tel samshjx@ sina com Received January Accepted March * Corresponding author Tel samshjx@ sina com

2 6 micrornas 541 micrornas and 744 mrna down-regulated at day 7 At day micrornas and 781 mrna were upregulated and 96 micrornas and 603 mrna down-regulated The RT-qPCR and Western blot confirmed that mir-29b were up-regulated while TGFβ3 expression down-regulated As mir-29b can interact with the 3'-UTR of TGFβ3 mrna to block its translation and TGFβ3 suppresses osteoblastic differentiation by either reduce Runx2 cellular protein and activity or induce to undergo Smurf-mediated Runx2 degradation thus lead to the promotion of osteoblastic differentiation The micrornas and genes changed during fibroblast osteogenic differentiation were mainly involved in BMPs Wnt and Ihh signal pathways suggesting that besides of some unique characteristics in the gene expression profile ligment fibroblasts might share a common pathway with osteoblasts during osteogenic differentiation Key words fibroblast osteogenic differentiation microrna mrna microarray heterotopic ossification HO DMEM GIBCO Trizol Invitrogen BCIP / NBT 1 Sigma Sigma β- Sigma 3 Sigma Ⅰ GIBCO RT-PCR 2 TOYOBO TGF-β3 Smad1 Proteintech RNeasy mini kit QIAGEN mirna RT-qPCR GeneCopoeia mir-31 * mir-29b GeneCopoeia PCR Ⅰ 1 cm 2 cm 3 cm PBS 4 NIH3T3 3 1 mm μg / ml Ⅰ dexamethasone 1 25 OH 2 D 3 3 h r / min 6 min d 5 d 1 Ⅰ / ml nmol / L 50 μg / ml β- 10 β- 95% 10 min 30 min d microrna mrna microrna mrna PCR real-time quantitative PCR RT-qPCR Western blotting BCIP / NBT 30 min 1 ~ % CO 2 mmol / L d d d PBS 3 95% 10 min d RT-PCR

3 Table 1 The sequences of PCR primers Gene Primer sequence Product length / bp Osteocalcin Runx2 GAPDH Forward 5'-ATGAGAGCCCTC ACACTCCTC-3' Reverse 5'-GCCGAAGCGCCGATAGGC-3' Forward 5'-CAGACCAGCAGCACTCCA-3' Reverse 5'-CAGACCAGCAGCACTCCA-3' Forward 5'-GGGCTGCTTTTAACTCTG-3' Reverse 5'-TGGCAGGTTTTTCTAGACGG-3' Runx2 Trizol normalization RNA RMA GAPDH PCR 95 5 min s s 72 1 min 34 Table 1 PCR 1% Runx2 1 3 microrna Trizol RNA RNeasy RNA 1 6 PCR Western mircury TM Array Power Hy3 TM / Hy5 TM mirna 31 * PCR RNeasy mini kit microrna CURY TM LNA Exiqon microrna mirbase mirplus TM Axon GenePix 4000B 3 3 GenePix pro V mrna Trizol RNA SDS-PAGE PVDF 1 h RNA cdna 4 TBS-T 3 10 min cdna 37 1 h TBS-T 3 10 min ECL Cy3 TGF-β3 Smad1 mrna 42 NimbleGen 60 mer NCBI 16 h Axon GenePix 4000B 2 1 GenePix pro V d TIFF NimbleScan Version2 5 mirna Fig 1 3 microrna mrna Foldchang 2 0 Agilent Gene Spring GX11 0 Gene Ontology GO Pathway mir-29b mir- microrna mir-29b mir-31 * 3' poly A poly A mirna 5 real-time PCR s s s Western TGF-β3 Smad1 Western 1 h d mrna NimbleScan RMA Robust Multichip Average Fig 2 quantile β-

4 6 micrornas 543 Fig 1 Morphological observation of ligament fibroblast under the light microscope 10 7 d 14 BCIP / NBT 7 d 14 d Fig 2 β- Runx2 14 d Fig 3 β- Fig 2 Effects of osteogenic medium on ligament fibroblast differentiation Ligament fibroblasts were stained for ALP expression analysis and Alizarin Red S were used for nodual mineralization detection after treating with osteogenic medium 10 nmol / L dexamethasone 50 μg / ml ascorbic acid and 10 mmol / L β-glycerophosphate for 7 days and 14 days Fig 3 Effect of osteogenic medium on the mrna expression of osteocalcin and Runx2 in ligament fibroblasts Ligament fibroblasts were treated with osteogenic medium 10 nmol / L dexamethasone 50 μg / ml ascorbic acid and 10 mmol / L β-glycerophosphate for 7 days and 14 days The mrna expression was examined by RT-PCR After osteogenic induction the mrna expression level of osteocalcin increased at 14 days but the expression level of Runx2 had no change 2 2 microrna 7 d 66 micrornas micrornas 94 micrornas micrornas 96 micrornas

5 mir-31 * 7 d 0 20 micrornas 14 d 0 13 Table 2 7 d mir-629 * 9 14 d Table 2 Difference expressed micrornas during fibroblast osteogenic differentiation Probes ID Name Fold change 7 d vs 0 d 14 d vs 0 d Regulation hsa-mir-629 * up hsa-mir-25 * up hsv1-mir-h6-3p up hsa-mirplus-f up hsa-mir-29b up hsa-mir-19a down hsa-mir down hsa-mir-127-3p down hsa-mir down hsa-mirplus-a down hsa-mir-574-5p down hsa-mir-31 * down hsa-mir down hsa-mir-24-1 * down hsa-let-7c down hsa-mir-103 N A 0 53 down N A Not available Fold change 1 30 or 0 77 P-Value mrna mrnas mrnas mrnas FOXF1 PDK4 FGF7 DKK1 ADRA2A ADAMTS14 7 d 640 Table 3 GO mrnas 744 mrnas TGF-β wnt 14 d 781 mrnas 603 Table 3 SEQ_ID Difference expressed mrna during fibroblast osteogenic differentiation Gene Fold change 7 d vs 0 d 14 d vs 0 d Regulation Gene Ontology GO BC FOXF up Transcription factor activity BC PDK up Phosphorylation AF MAP4K up Protein amino acid phosphorylation BC IL1RL up Interleukin-1 receptor activity BC FGF up Growth factor activity AF ADAMTS down Collagen fibril organization BC ADRA2A down Activation of MAPK activity AB COL16A down Phosphate transport NM_ COL15A down Cell differentiation BC DKK down Wnt receptor signaling pathway Fold change 2 0 P-value PCR Western PCR 7 d mir-29b 4 34 mir-31 * d mir-29b 4 51 mirβ- 31 * 0 13 Fig 4 Western

6 6 micrornas 545 TGFβ3 smad1 β- Fig 5 Fig 4 Comparison of RT-qPCR and microarray on the microrna expression level of ligament fibroblast treated with osteogenic medium for 7 days and 14 days The results of RT-qPCR were consistent with the microarray The black columns represented the relative levels of microrna from microarray and gray columns represented the relative levels from RT-qPCR Fig 5 Expression levels of microrna-target genes smad1 and TGFβ3 were examined by Western blotting Ligament fibroblast were treated with osteogenic medium 10 nmol / L dexamethasone 50 μg / ml ascorbic acid and 10 mmol / L β-glycerophosphate for 7 day and 14 day and then the levels of smad1 and TGFβ3 were determined by Western blot analysis using total protein lysates 3 Williams Runx2 Smurf Runx2 9 qrt-pcr mir-29b Ⅰ Western TGF-β3 TGF-β3 mir-29b mir-29b TGFβ3 Runx2 β- 29b TGF-β3 ACTVR2A COL1A1 8 TGFβ Smad3 mrnas FOXF1 PRRG4 MAP4K4 DKK1 Runx2 ADRA2A ADAMTS14 GO microrna mrna BMPs Wnt Hedgehog microrna DKK1 Wnt mir-29b Wnt 8 FOXF1 Hedgehog mir- Hedgehog 10

7 BMPs Wnt Hedgehog ACVR1 Smad1 SATB2 Smad1 BMP microrna mrna mir-31 * Western blot Smad1 microrna mir-15a mir-629 * mir-101 mir-31 * mir-744 mir-98 microrna BMP BMP mir-133 mir-135 mir-31 * mir PRRG4 mir-101 mir-93 microrna microrna 2 microrna PPP1R15B LAMP2 DUSP16 mir-145 LRP2 MAP4K4 BMP3 Table 4 13 MicroRNA mrna mir microrna mrna 1 mrna microrna microrna mrna microrna mir-145 MAP4K4 mtor MiRNA γ receptor 1 mirna peroxisome proliferator-activated receptor γ PPARγ mirna 1 PPARγ 1 16 mirna mir-98 DUSP16 14 microrna GO MAPK mrna microrna org-targets and expression http / / www microrna org / TargetScanHuman 5 1 http / / www targetscan org / mir-31 * Table 4 mirna mir-15a Smad1 Smad4 DNA mir-17 microrna mir-98 PRRG4 Wnt Hedgehog Putative targets of significantly changed micrornas in ligament fibroblast osteogenic differentiation Putative targets WNT3A FGF7 Dlk1 BCL-2 Smad7 ACVR2A IRAK2 IL10RA IHH WNT4 CACNA1E BMPR1A IGF2R IL1RAPL1 et al mir-25 * mir-31 * mir-145 mir-574-5p mir-19a mir-103 Smad3 KLF4 MAP2K4 CACNA1I COL1A2 BMPR2 TGIF1 Smad7 Cbfa2T3 GDF11 NF1 CRTAM CALM3 et al ACVR1 SMAD1 SATB2 Dlx3 FGF7 FGF10 CD28 MAP3K1 COL5A1 PPP1R9A et al Smad3 Smad5 TNFRSF11B FGF5 CACNB2 CAMK1D ACVR2A TGFBR2 CAMK2D Smad4 RUNX3 BMP3 MAP2K4 ESR1 CABP1 IGF1R FXR1 LRP2 MAP4K4 et al CALCOCO1 IHH HIF3A Smad5 SERP1 CALCOCO1 CD96 CACNA2D2 CTNND2 TCF20 RFX4 CALML4 et al BMPER DKK3 RUNX3 SATB1 SMAD4 TNFRSF1B STAT2 SMAD5 WNT1 TNFRSF12A CBFA2T2 TGFBR2 BMP3 CACNA1C BMPR2 et al DKK1 CAMKK1 FZD4 NOTCH2 TGIF2 RUNX1T1 LRP2 RCAN1 ESR1 WNT16 et al

8 6 micrornas 547 References 1 Wilkinson J M Stockley I Hamer A J et al Biochemical markers of bone turnover and development of heterotopic ossification after total hip arthroplasty J J Orthop Res Tam L S Gu J Yu D Pathogenesis of ankylosing spondylitis J Nat Rev Rheumatol Ducy P Schinke T Karsenty G The osteoblast a sophisticated fibroblast under central surveillance J Science mirnas 4 J Wang Yan Wu Qiang Hu J Wang Xiao-Feng Gao Gen-De Xiang-Yang et al Expression profiles ofm irnas relative to the The study of fibroblasts from ankylosig spondylitis secrete osteocalcin in vitro J Orthop J China Shui C Scutt A M Mouse embryo-derived NIH3T3 fibroblasts adopt an osteoblast-like phenotype when treated with 1alpha 25- dihydroxyvitamin D 3 and dexamethasone in vitro J J Cell Physiol Williams D C Boder G B Toomey R E et al Mineralization and metabolic response in serially passaged adult rat bone cells J Calcif Tissue Int J Gao Gen- Pan Qiu-Hui Li Yi-Guang Yang Song-Hai et al Bone De Liu Yao-Sheng Osteogenic role played by fibroblasts of the supra-interspinous ligament of ankylosing spondylitins in vitro morphogenetic protein 2 up regulates osterix expression through Smad pathway J Chin J Biochem Mol Biol J Chin J Rheumatol Li Z Hassan M Q Jafferji M et al Biological functions of mir- 29b contribute to positive regulation of osteoblast differentiation J J Biol Chem Kang J S Alliston T Delston R et al Repression of Runx2 function by TGF-beta through recruitment of class Ⅱ histone deacetylases by Smad3 J EMBO J Wendling D S Luck C von Schweinitz D et al Characteristic overexpression of the forkhead box transcription factor Foxf1 in patched-associated tumors J Int J Mol Med Li Z Hassan M Q Volinia S et al A microrna signature for a BMP2-induced osteoblast lineage commitment program J Proc Natl Acad Sci U S A Huang J Zhao L Xing L et al MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation J Stem Cells invasiveness of breast cancer cell J Acta Univ Med Anhui microrna J Li Guo-Xing Ning Xiao-Min Kang Xiang-Tao et al MicroRNAs regulate animals adipocyte differentiation J Chin J Biochem Mol Biol Smad Osterix J Guntur K V Guilherme A Xue L et al Map4k4 negatively regulates peroxisome proliferator-activated receptor PPAR gamma protein translation by suppressing the mammalian target of rapamycin mtor signaling pathway in cultured adipocytes J J Biol Chem