In Vitro Cellular & Developmental Biology - Animal

, Volume 51, Issue 8, pp 797–807 | Cite as

Integrated miRNA and mRNA expression profiling of tension force-induced bone formation in periodontal ligament cells

  • Maolin Chang
  • Heng Lin
  • Meng Luo
  • Jie Wang
  • Guangli HanEmail author


Tension force-induced bone formation is a complex biological process altered by various factors, for example miRNAs and gene regulatory network. However, we know little about critical gene regulators and their functional consequences on this complex process. The aim of this study was to determine the integrated relation between microRNA and mRNA expression in tension force-induced bone formation in periodontal ligament cells by a system biological approach. We identified 818 mRNAs and 32 miRNAs differentially expressed between cyclic tension force-stimulated human periodontal ligament cells and control cells by microarrays. By using miRNA/mRNA network analysis, protein-protein interactions network analysis, and hub analysis, we found that miR-195-5p, miR-424-5p, miR-1297, miR-3607-5p, miR-145-5p, miR-4328, and miR-224-5p were core microRNAs of tension force-induced bone formation. WDR33, HSPH1, ERBB3, RIF1, IKBKB, CREB1, FGF2, and PAG1 were identified as hubs of the PPI network, suggesting the biological significance in this process. The miRNA expression was further examined in human PDLC and animal samples by using quantitative real-time PCR. Thus, we proposed a model of tension force-induced bone formation which is co-regulated through integration of the miRNA and mRNA. This study illustrated the benefits of system biological approaches in the analysis of tension force-induced bone formation as a complex biological process. We used public information and our experimental data to do comprehensive analysis and revealed the coordination transcriptional control of miRNAs of tension force-induced bone formation.


MiRNA Gene expression profiling Bone formation Network Orthodontic tooth movement 



This study was supported by the National Natural Science Foundation of China (NSFC); NSFC number: 81371169.

Supplementary material

11626_2015_9892_MOESM1_ESM.xls (3.4 mb)
ESM 1 (XLS 3517 kb)
11626_2015_9892_MOESM2_ESM.xls (56 kb)
ESM 2 (XLS 56 kb)
11626_2015_9892_Fig6_ESM.jpg (1.2 mb)
Supplementary Fig. 1

Description of in vivo orthodontic force applying system. (a) Mice positioned in the supine position on a surgical table. (b) Surgical table placed under a stereomicroscope with light system. (c) Use of a mouth-opener in a mouse. (d) Occlusal view of the maxillary molar region. The distal end of a nickel–titanium open-coil spring (Tomy, Japan) was bonded to the occlusal surface of the right first maxillary molar using a light-cured resin. (e) A tension gauge was attached to the surgical table to show the force magnitude. (f) The coil spring bonded to both upper incisors. Stereomicroscope (S), mouth-opener (O), coil spring (C), upper first molar (M), tension gauge (T). (JPEG 1223 kb)

11626_2015_9892_Fig7_ESM.jpg (354 kb)
Supplementary Fig. 2

Validation of differentially expressed mRNAs in vivo. (a-h) The mRNAs expression of WDR33, RIF1, IKBKB, CREB1, FGF2, PAG1 and HSPH1 at tension sites of orthodontic tooth movement mice molar at day 3 was determined by real-time PCR. β-actin was used as a normalization control. All data were based on three independent experiments. Asterisks indicate statistical significance between experimental and control groups at P < 0.05. (JPEG 354 kb)

11626_2015_9892_Fig8_ESM.jpg (915 kb)
Supplementary Fig. 3

Strongly expression of FGF2 and CREB1 at tension sites of orthodontic tooth movement mice molar. (a) FGF2 expression was detected by immunofluorescence staining. FGF2 was strongly localized at tension site of first molar but were weakly localized at the contralateral sites at day 3. TS: tension site; CS: contralateral site. (Original magnification, 200×) (b) CREB1 expression was detected by immunohistochemistry staining. CREB1 was strongly localized at tension sites of first molar but were weakly localized at the compression sites at day 3. Scale bar for A, 200 μm; scale bar for B and C, 100 μm. TS: tension site; CS: compression site. (JPEG 914 kb)


  1. Byun MR, Kim AR, Hwang JH, Kim KM, Hwang ES, Hong JH (2014) FGF2 stimulates osteogenic differentiation through ERK induced TAZ expression. Bone 58:72–80CrossRefPubMedGoogle Scholar
  2. Chen L, Holmstrom K, Qiu W, Ditzel N, Shi K, Hokland L, Kassem M (2014) MicroRNA-34a inhibits osteoblast differentiation and in vivo bone formation of human stromal stem cells. Stem Cells 32(4):902–912CrossRefPubMedGoogle Scholar
  3. Finnerty JR, Wang WX, Hebert SS, Wilfred BR, Mao G, Nelson PT (2010) The miR-15/107 group of microRNA genes: evolutionary biology, cellular functions, and roles in human diseases. J Mol Biol 402(3):491–509PubMedCentralCrossRefPubMedGoogle Scholar
  4. Friedman RC, Farh KKH, Burge CB, Bartel DP (2009) Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 19(1):92–105PubMedCentralCrossRefPubMedGoogle Scholar
  5. Gao J, Yang T, Han J, Yan K, Qiu X, Zhou Y, Fan Q, Ma B (2011) MicroRNA expression during osteogenic differentiation of human multipotent mesenchymal stromal cells from bone marrow. J Cell Biochem 112(7):1844–1856CrossRefPubMedGoogle Scholar
  6. Gay I, Cavender A, Peto D, Sun Z, Speer A, Cao H, Amendt BA (2014) Differentiation of human dental stem cells reveals a role for microRNA-218. J Periodontal Res 49(1):110–120PubMedCentralCrossRefPubMedGoogle Scholar
  7. He JF, Luo YM, Wan XH, Jiang D (2011) Biogenesis of MiRNA-195 and its role in biogenesis, the cell cycle, and apoptosis. J Biochem Mol Toxicol 25(6):404–408CrossRefPubMedGoogle Scholar
  8. He X, Zhang J (2006) Why do hubs tend to be essential in protein networks? PLoS Genet 2(6):e88PubMedCentralCrossRefPubMedGoogle Scholar
  9. Huang XF, Zhao YB, Zhang FM, Han PY (2009) Comparative study of gene expression during tooth eruption and orthodontic tooth movement in mice. Oral Dis 15(8):573–579CrossRefPubMedGoogle Scholar
  10. Hung PS, Chen FC, Kuang SH, Kao SY, Lin SC, Chang KW (2010) miR-146a induces differentiation of periodontal ligament cells. J Dent Res 89(3):252–257CrossRefPubMedGoogle Scholar
  11. Jia J, Tian Q, Ling S, Liu Y, Yang S, Shao Z (2013) miR-145 suppresses osteogenic differentiation by targeting Sp7. FEBS Lett 587(18):3027–3031CrossRefPubMedGoogle Scholar
  12. Krishnan V, Davidovitch Z (2006) Cellular, molecular, and tissue-level reactions to orthodontic force. Am J Orthod Dentofac Orthop 129(4):469, e461–432CrossRefGoogle Scholar
  13. Ku SJ, Chang YI, Chae CH, Kim SG, Park YW, Jung YK, Choi JY (2009) Static tensional forces increase osteogenic gene expression in three-dimensional periodontal ligament cell culture. BMB Rep 42(7):427–432CrossRefPubMedGoogle Scholar
  14. Li C, Bai Y, Liu H, Zuo X, Yao H, Xu Y, Cao M (2013) Comparative study of microRNA profiling in keloid fibroblast and annotation of differential expressed microRNAs. Acta Biochim Biophys Sin 45(8):692–699Google Scholar
  15. Li C, Li C, Yue J, Huang X, Chen M, Gao J, Wu B (2012) miR-21 and miR-101 regulate PLAP-1 expression in periodontal ligament cells. Mol Med Rep 5(5):1340–1346PubMedGoogle Scholar
  16. Liu H, Lin H, Zhang L, Sun Q, Yuan G, Zhang L, Chen S, Chen Z (2013) miR-145 and miR-143 regulate odontoblast differentiation through targeting Klf4 and Osx genes in a feedback loop. J Biol Chem 288(13):9261–9271PubMedCentralCrossRefPubMedGoogle Scholar
  17. Lin SH, Cheng CJ, Lee YC, Ye X, Tsai WW, Kim J, Pasqualini R, Arap W, Navone NM, Tu SM, Hu M, Yu-Lee LY, Logothetis CJ (2008) A 45-kDa ErbB3 secreted by prostate cancer cells promotes bone formation. Oncogene 27(39):5195–5203PubMedCentralCrossRefPubMedGoogle Scholar
  18. Liu Y, Liu W, Hu C, Xue Z, Wang G, Ding B, Luo H, Tang L, Kong X, Chen X, Liu N, Ding Y, Jin Y (2011) MiR-17 modulates osteogenic differentiation through a coherent feed-forward loop in mesenchymal stem cells isolated from periodontal ligaments of patients with periodontitis. Stem Cells 29(11):1804–1816CrossRefPubMedGoogle Scholar
  19. Matsubara T, Ikeda F, Hata K, Nakanishi M, Okada M, Yasuda H, Nishimura R, Yoneda T (2010) Cbp recruitment of Csk into lipid rafts is critical to c-Src kinase activity and bone resorption in osteoclasts. J Bone Miner Res 25(5):1068–1076PubMedGoogle Scholar
  20. Mosakhani N, Pazzaglia L, Benassi MS, Borze I, Quattrini I, Picci P, Knuutila S (2013) MicroRNA expression profiles in metastatic and non-metastatic giant cell tumor of bone. Histol Histopathol 28(5):671–678PubMedGoogle Scholar
  21. Namlos HM, Meza-Zepeda LA, Baroy T, Ostensen IH, Kresse SH, Kuijjer ML, Serra M, Burger H, Cleton-Jansen AM, Myklebost O (2012) Modulation of the osteosarcoma expression phenotype by microRNAs. PLoS ONE 7(10):e48086PubMedCentralCrossRefPubMedGoogle Scholar
  22. Otero JE, Dai S, Alhawagri MA, Darwech I, Abu-Amer Y (2010) IKKbeta activation is sufficient for RANK-independent osteoclast differentiation and osteolysis. J Bone Miner Res 25(6):1282–1294PubMedCentralCrossRefPubMedGoogle Scholar
  23. Palmieri A, Pezzetti F, Brunelli G, Martinelli M, Lo Muzio L, Scarano A, Degidi M, Piattelli A, Carinci F (2008) Peptide-15 changes miRNA expression in osteoblast-like cells. Implant Dent 17(1):100–108CrossRefPubMedGoogle Scholar
  24. Pinkerton MN, Wescott DC, Gaffey BJ, Beggs KT, Milne TJ, Meikle MC (2008) Cultured human periodontal ligament cells constitutively express multiple osteotropic cytokines and growth factors, several of which are responsive to mechanical deformation. J Periodontal Res 43(3):343–351CrossRefPubMedGoogle Scholar
  25. Saini S, Majid S, Shahryari V, Tabatabai ZL, Arora S, Yamamura S, Tanaka Y, Dahiya R, Deng G (2014) Regulation of SRC kinases by microRNA-3607 located in a frequently deleted locus in prostate cancer. Mol Cancer Ther 13(7):1952–1963PubMedCentralCrossRefPubMedGoogle Scholar
  26. Schaap-Oziemlak AM, Raymakers RA, Bergevoet SM, Gilissen C, Jansen BJ, Adema GJ, Kogler G, le Sage C, Agami R, van der Reijden BA, Jansen JH (2010) MicroRNA hsa-miR-135b regulates mineralization in osteogenic differentiation of human unrestricted somatic stem cells. Stem Cells Dev 19(6):877–885CrossRefPubMedGoogle Scholar
  27. Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N (2008) Widespread changes in protein synthesis induced by microRNAs. Nature 455(7209):58–63CrossRefPubMedGoogle Scholar
  28. Song X, Liu W, Xie S, Wang M, Cao G, Mao C, Lv C (2013) All-transretinoic acid ameliorates bleomycin-induced lung fibrosis by downregulating the TGF-beta1/Smad3 signaling pathway in rats. Lab Investig 93(11):1219–1231CrossRefPubMedGoogle Scholar
  29. Taddei SR, Moura AP, Andrade I Jr, Garlet GP, Garlet TP, Teixeira MM, da Silva TA (2012) Experimental model of tooth movement in mice: a standardized protocol for studying bone remodeling under compression and tensile strains. J Biomech 45(16):2729–2735CrossRefPubMedGoogle Scholar
  30. Wei FD, Liu C, Feng F, Zhang S, Yang Y, Hu G Ding and Wang S (2014) MicroRNA-21 mediates stretch-induced osteogenic differentiation in human periodontal ligament stem cells. Stem Cells DevGoogle Scholar
  31. Wescott D, Pinkerton M, Gaffey B, Beggs K, Milne T, Meikle M (2007) Osteogenic gene expression by human periodontal ligament cells under cyclic tension. J Dent Res 86(12):1212–1216CrossRefPubMedGoogle Scholar
  32. Wise GE, King GJ (2008) Mechanisms of tooth eruption and orthodontic tooth movement. J Dent Res 87(5):414–434PubMedCentralCrossRefPubMedGoogle Scholar
  33. Yamaguchi M, Shimizu N, Shibata Y, Abiko Y (1996) Effects of different magnitudes of tension-force on alkaline phosphatase activity in periodontal ligament cells. J Dent Res 75(3):889–894CrossRefPubMedGoogle Scholar
  34. Yang NQ, Zhang J, Tang QY, Guo JM, Wang GM (2014) miRNA-1297 induces cell proliferation by targeting phosphatase and Tensin homolog in testicular germ cell tumor cells. Asian Pac J Cancer Prev 15(15):6243–6246CrossRefPubMedGoogle Scholar
  35. Yang Y, Li X, Rabie A, Fu M, Zhang D (2005) Human periodontal ligament cells express osteoblastic phenotypes under intermittent force loading in vitro. Front Biosci J Virtual Libr 11:776–781CrossRefGoogle Scholar
  36. Yang YQ, Li XT, Rabie AB, Fu MK, Zhang D (2006) Human periodontal ligament cells express osteoblastic phenotypes under intermittent force loading in vitro. Front Biosci 11:776–781CrossRefPubMedGoogle Scholar
  37. Zainal Ariffin SH, Yamamoto Z, Zainol Abidin IZ, Megat Abdul Wahab R, Zainal Ariffin Z (2011) Cellular and molecular changes in orthodontic tooth movement. ScientificWorldJournal 11:1788–1803PubMedCentralCrossRefPubMedGoogle Scholar
  38. Zhang RJ, Edwards R, Ko SY, Dong S, Liu H, Oyajobi BO, Papasian C, Deng HW, Zhao M (2011) Transcriptional regulation of BMP2 expression by the PTH-CREB signaling pathway in osteoblasts. PLoS ONE 6(6):e20780PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2015

Authors and Affiliations

  • Maolin Chang
    • 1
  • Heng Lin
    • 1
  • Meng Luo
    • 1
  • Jie Wang
    • 1
  • Guangli Han
    • 1
    Email author
  1. 1.State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education (KLOBM), School and Hospital of StomatologyWuhan UniversityWuhanChina

Personalised recommendations