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Regulation of osteogenesis via miR-101-3p in mesenchymal stem cells by human gingival fibroblasts

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Abstract

Introduction

Mesenchymal stem cells (MSCs) can differentiate into various types of cells and can thus be used for periodontal regenerative therapy. However, the mechanism of differentiation is still unclear. Transplanted MSCs are, via their transcription factors or microRNAs (miRNAs), affected by periodontal cells with direct contact or secretion of humoral factors. Therefore, transplanted MSCs are regulated by humoral factors from human gingival fibroblasts (HGF). Moreover, insulin-like growth factor (IGF)-1 is secreted from HGF and regulates periodontal regeneration. To clarify the regulatory mechanism for MSC differentiation by humoral factors from HGF, we identified key genes, specifically miRNAs, involved in this process, and determined their function in MSC differentiation.

Materials and Methods

Mesenchymal stem cells were indirectly co-cultured with HGF in osteogenic or growth conditions and then evaluated for osteogenesis, undifferentiated MSC markers, and characteristic miRNAs. MSCs had their miRNA expression levels adjusted or were challenged with IGF-1 during osteogenesis, or both of which were performed, and then, MSCs were evaluated for osteogenesis or undifferentiated MSC markers.

Results

Mesenchymal stem cells co-cultured with HGF showed suppression of osteogenesis and characteristic expression of ETV1, an undifferentiated MSC marker, as well as miR-101-3p. Over-expression of miR-101-3p regulated osteogenesis and ETV1 expression as well as indirect co-culture with HGF. IGF-1 induced miR-101-3p and ETV1 expression. However, IGF-1 did not suppress osteogenesis.

Conclusions

Humoral factors from HGF suppressed osteogenesis in MSCs. The effect was regulated by miRNAs and undifferentiated MSC markers. miR-101-3p and ETV1 were the key factors and were regulated by IGF-1.

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References

  1. Nagatomo K, Komaki M, Sekiya I, Sakaguchi Y, Noguchi K, Oda S, Muneta T, Ishikawa I (2006) Stem cell properties of human periodontal ligament cells (in eng). J Periodontal Res 41:303–310. https://doi.org/10.1111/j.1600-0765.2006.00870.x

    Article  CAS  PubMed  Google Scholar 

  2. Seo BM, Miura M, Gronthos S, Bartold PM, Batouli S, Brahim J, Young M, Robey PG, Wang CY, Shi S (2004) Investigation of multipotent postnatal stem cells from human periodontal ligament (in eng). Lancet (London, England) 364:149–155. https://doi.org/10.1016/s0140-6736(04)16627-0

    Article  CAS  Google Scholar 

  3. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells (in eng). Science (New York, NY) 284:143–147

    Article  CAS  Google Scholar 

  4. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM (2002) Pluripotency of mesenchymal stem cells derived from adult marrow (in eng). Nature 418:41–49. https://doi.org/10.1038/nature00870

    Article  CAS  Google Scholar 

  5. Hasegawa N, Kawaguchi H, Hirachi A, Takeda K, Mizuno N, Nishimura M, Koike C, Tsuji K, Iba H, Kato Y, Kurihara H (2006) Behavior of transplanted bone marrow-derived mesenchymal stem cells in periodontal defects (in eng). J Periodontol 77:1003–1007. https://doi.org/10.1902/jop.2006.050341

    Article  PubMed  Google Scholar 

  6. Kawaguchi H, Hirachi A, Hasegawa N, Iwata T, Hamaguchi H, Shiba H, Takata T, Kato Y, Kurihara H (2004) Enhancement of periodontal tissue regeneration by transplantation of bone marrow mesenchymal stem cells (in eng). J Periodontol 75:1281–1287. https://doi.org/10.1902/jop.2004.75.9.1281

    Article  PubMed  Google Scholar 

  7. Kittaka M, Kajiya M, Shiba H, Takewaki M, Takeshita K, Khung R, Fujita T, Iwata T, Nguyen TQ, Ouhara K, Takeda K, Fujita T, Kurihara H (2015) Clumps of a mesenchymal stromal cell/extracellular matrix complex can be a novel tissue engineering therapy for bone regeneration (in eng). Cytotherapy 17:860–873. https://doi.org/10.1016/j.jcyt.2015.01.007

    Article  CAS  PubMed  Google Scholar 

  8. Mizuno N, Ozeki Y, Shiba H, Kajiya M, Nagahara T, Takeda K, Kawaguchi H, Abiko Y, Kurihara H (2008) Humoral factors released from human periodontal ligament cells influence calcification and proliferation in human bone marrow mesenchymal stem cells (in eng). J Periodontol 79:2361–2370. https://doi.org/10.1902/jop.2008.070577

    Article  CAS  PubMed  Google Scholar 

  9. Zhou Y, Zimber M, Yuan H, Naughton GK, Fernan R, Li WJ (2016) Effects of human fibroblast-derived extracellular matrix on mesenchymal stem cells (in eng). Stem Cell Rev Rep 12:560–572. https://doi.org/10.1007/s12015-016-9671-7

    Article  CAS  PubMed  Google Scholar 

  10. Wu Y, Peng Y, Gao D, Feng C, Yuan X, Li H, Wang Y, Yang L, Huang S, Fu X (2015) Mesenchymal stem cells suppress fibroblast proliferation and reduce skin fibrosis through a TGF-beta3-dependent activation (in eng). Int J Low Extrem Wounds 14:50–62. https://doi.org/10.1177/1534734614568373

    Article  CAS  Google Scholar 

  11. Nakayama Y, Takai H, Matsui S, Zhou L, Abiko Y, Ganss B, Ogata Y (2014) Transcriptional regulation of amelotin gene by proinflammatory cytokines in gingival fibroblasts (in eng). Connect Tissue Res 55:18–20. https://doi.org/10.3109/03008207.2014.923848

    Article  CAS  PubMed  Google Scholar 

  12. Ogata Y, Matsui S, Kato A, Zhou L, Nakayama Y, Takai H (2014) MicroRNA expression in inflamed and noninflamed gingival tissues from Japanese patients (in eng). J Oral Sci 56:253–260

    Article  CAS  Google Scholar 

  13. Ishii M, Koike C, Igarashi A, Yamanaka K, Pan H, Higashi Y, Kawaguchi H, Sugiyama M, Kamata N, Iwata T, Matsubara T, Nakamura K, Kurihara H, Tsuji K, Kato Y (2005) Molecular markers distinguish bone marrow mesenchymal stem cells from fibroblasts (in eng). Biochem Biophys Res Commun 332:297–303. https://doi.org/10.1016/j.bbrc.2005.04.118

    Article  CAS  PubMed  Google Scholar 

  14. Kubo H, Shimizu M, Taya Y, Kawamoto T, Michida M, Kaneko E, Igarashi A, Nishimura M, Segoshi K, Shimazu Y, Tsuji K, Aoba T, Kato Y (2009) Identification of mesenchymal stem cell (MSC)-transcription factors by microarray and knockdown analyses, and signature molecule-marked MSC in bone marrow by immunohistochemistry (in eng). Genes Cells 14:407–424. https://doi.org/10.1111/j.1365-2443.2009.01281.x

    Article  CAS  PubMed  Google Scholar 

  15. Kapinas K, Kessler C, Ricks T, Gronowicz G, Delany AM (2010) miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop (in eng). J Biol Chem 285:25221–25231. https://doi.org/10.1074/jbc.M110.116137

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Huang J, Zhao L, Xing L, Chen D (2010) MicroRNA-204 regulates Runx2 protein expression and mesenchymal progenitor cell differentiation (in eng). Stem Cells (Dayton, Ohio) 28:357–364. https://doi.org/10.1002/stem.288

    Article  CAS  Google Scholar 

  17. Tome M, Lopez-Romero P, Albo C, Sepulveda JC, Fernandez-Gutierrez B, Dopazo A, Bernad A, Gonzalez MA (2011) miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells (in eng). Cell Death Differ 18:985–995. https://doi.org/10.1038/cdd.2010.167

    Article  CAS  PubMed  Google Scholar 

  18. Qiu Y, Chen Y, Zeng T, Guo W, Zhou W, Yang X (2016) EGCG ameliorates the hypoxia-induced apoptosis and osteogenic differentiation reduction of mesenchymal stem cells via upregulating miR-210 (in eng). Mol Biol Rep 43:183–193. https://doi.org/10.1007/s11033-015-3936-0

    Article  CAS  PubMed  Google Scholar 

  19. Jones JI, Clemmons DR (1995) Insulin-like growth factors and their binding proteins: biological actions (in eng). Endocr Rev 16:3–34. https://doi.org/10.1210/edrv-16-1-3

    Article  CAS  PubMed  Google Scholar 

  20. Saygun I, Karacay S, Serdar M, Ural AU, Sencimen M, Kurtis B (2008) Effects of laser irradiation on the release of basic fibroblast growth factor (bFGF), insulin like growth factor-1 (IGF-1), and receptor of IGF-1 (IGFBP3) from gingival fibroblasts (in eng). Lasers Med Sci 23:211–215. https://doi.org/10.1007/s10103-007-0477-3

    Article  PubMed  Google Scholar 

  21. Ochiai H, Okada S, Saito A, Hoshi K, Yamashita H, Takato T, Azuma T (2012) Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-beta1 (TGF-beta1) administration suppresses osteoblast differentiation (in eng). J Biol Chem 287:22654–22661. https://doi.org/10.1074/jbc.M111.279091

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Liu GX, Ma S, Li Y, Yu Y, Zhou YX, Lu YD, Jin L, Wang ZL, Yu JH (2018) Hsa-let-7c controls the committed differentiation of IGF-1-treated mesenchymal stem cells derived from dental pulps by targeting IGF-1R via the MAPK pathways (in eng). Exp Mol Med 50:25. https://doi.org/10.1038/s12276-018-0048-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Iwata T, Kawamoto T, Sasabe E, Miyazaki K, Fujimoto K, Noshiro M, Kurihara H, Kato Y (2006) Effects of overexpression of basic helix-loop-helix transcription factor Dec1 on osteogenic and adipogenic differentiation of mesenchymal stem cells (in eng). Eur J Cell Biol 85:423–431. https://doi.org/10.1016/j.ejcb.2005.12.007

    Article  CAS  PubMed  Google Scholar 

  24. Lindhe J, Pontoriero R, Berglundh T, Araujo M (1995) The effect of flap management and bioresorbable occlusive devices in GTR treatment of degree III furcation defects. An experimental study in dogs (in eng). J Clin Periodontol 22:276–283

    Article  CAS  Google Scholar 

  25. Dighe AS, Yang S, Madhu V, Balian G, Cui Q (2013) Interferon gamma and T cells inhibit osteogenesis induced by allogeneic mesenchymal stromal cells (in eng). J Orthop Res 31:227–234. https://doi.org/10.1002/jor.22212

    Article  CAS  PubMed  Google Scholar 

  26. Yang N, Wang G, Hu C, Shi Y, Liao L, Shi S, Cai Y, Cheng S, Wang X, Liu Y, Tang L, Ding Y, Jin Y (2013) Tumor necrosis factor alpha suppresses the mesenchymal stem cell osteogenesis promoter miR-21 in estrogen deficiency-induced osteoporosis (in eng). J Bone Miner Res 28:559–573. https://doi.org/10.1002/jbmr.1798

    Article  CAS  Google Scholar 

  27. Ciciarello M, Zini R, Rossi L, Salvestrini V, Ferrari D, Manfredini R, Lemoli RM (2013) Extracellular purines promote the differentiation of human bone marrow-derived mesenchymal stem cells to the osteogenic and adipogenic lineages (in eng). Stem Cells Dev 22:1097–1111. https://doi.org/10.1089/scd.2012.0432

    Article  CAS  Google Scholar 

  28. Suzuki K, Onoe K, Takahira H (1992) Activation of Ca(2+)-dependent K+ channel and Cl- conductance in canine pancreatic acinar cells through a cyclic AMP pathway (in eng). Jpn J Physiol 42:267–281

    Article  CAS  Google Scholar 

  29. Asami S, Chin M, Shichino H, Yoshida Y, Nemoto N, Mugishima H, Suzuki T (2008) Treatment of Ewing's sarcoma using an antisense oligodeoxynucleotide to regulate the cell cycle (in eng). Biol Pharm Bull 31:391–394

    Article  CAS  Google Scholar 

  30. Okazawa M, Abe H, Nakanishi S (2016) The Etv1 transcription factor activity-dependently downregulates a set of genes controlling cell growth and differentiation in maturing cerebellar granule cells (in eng). Biochem Biophys Res Commun 473:1071–1077. https://doi.org/10.1016/j.bbrc.2016.04.017

    Article  CAS  PubMed  Google Scholar 

  31. Adams KL, Rousso DL, Umbach JA, Novitch BG (2015) Foxp1-mediated programming of limb-innervating motor neurons from mouse and human embryonic stem cells (in eng). Nat Commun 6:6778. https://doi.org/10.1038/ncomms7778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li H, Liu P, Xu S, Li Y, Dekker JD, Li B, Fan Y, Zhang Z, Hong Y, Yang G, Tang T, Ren Y, Tucker HO, Yao Z, Guo X (2017) FOXP1 controls mesenchymal stem cell commitment and senescence during skeletal aging (in eng). J Clin Investig 127:1241–1253. https://doi.org/10.1172/jci89511

    Article  PubMed  Google Scholar 

  33. Park IH, Kim KH, Choi HK, Shim JS, Whang SY, Hahn SJ, Kwon OJ, Oh IH (2013) Constitutive stabilization of hypoxia-inducible factor alpha selectively promotes the self-renewal of mesenchymal progenitors and maintains mesenchymal stromal cells in an undifferentiated state (in eng). Exp Mol Med 45:e44. https://doi.org/10.1038/emm.2013.87

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Foshay KM, Gallicano GI (2007) Small RNAs, big potential: the role of MicroRNAs in stem cell function (in eng). Curr Stem Cell Res Ther 2:264–271

    Article  CAS  Google Scholar 

  35. Gits CM, van Kuijk PF, Jonkers MB, Boersma AW, van Ijcken WF, Wozniak A, Sciot R, Rutkowski P, Schoffski P, Taguchi T, Mathijssen RH, Verweij J, Sleijfer S, Debiec-Rychter M, Wiemer EA (2013) MiR-17-92 and miR-221/222 cluster members target KIT and ETV1 in human gastrointestinal stromal tumours (in eng). Br J Cancer 109:1625–1635. https://doi.org/10.1038/bjc.2013.483

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Li J, Lai Y, Ma J, Liu Y, Bi J, Zhang L, Chen L, Yao C, Lv W, Chang G, Wang S, Ouyang M, Wang W (2017) miR-17-5p suppresses cell proliferation and invasion by targeting ETV1 in triple-negative breast cancer (in eng). BMC Cancer 17:745. https://doi.org/10.1186/s12885-017-3674-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Isenmann S, Arthur A, Zannettino AC, Turner JL, Shi S, Glackin CA, Gronthos S (2009) TWIST family of basic helix-loop-helix transcription factors mediate human mesenchymal stem cell growth and commitment (in eng). Stem Cells (Dayton, Ohio) 27:2457–2468. https://doi.org/10.1002/stem.181

    Article  CAS  Google Scholar 

  38. Chang J, Liu F, Lee M, Wu B, Ting K, Zara JN, Soo C, Al Hezaimi K, Zou W, Chen X, Mooney DJ, Wang CY (2013) NF-kappaB inhibits osteogenic differentiation of mesenchymal stem cells by promoting beta-catenin degradation (in eng). Proc Natl Acad Sci USA 110:9469–9474. https://doi.org/10.1073/pnas.1300532110

    Article  Google Scholar 

  39. Matsubara T, Tsutsumi S, Pan H, Hiraoka H, Oda R, Nishimura M, Kawaguchi H, Nakamura K, Kato Y (2004) A new technique to expand human mesenchymal stem cells using basement membrane extracellular matrix (in eng). Biochem Biophys Res Commun 313:503–508

    Article  CAS  Google Scholar 

  40. Kaewsrichan J, Wongwitwichot P, Chandarajoti K, Chua KH, Ruszymah BH (2011) Sequential induction of marrow stromal cells by FGF2 and BMP2 improves their growth and differentiation potential in vivo (in eng). Arch Oral Biol 56:90–101. https://doi.org/10.1016/j.archoralbio.2010.09.003

    Article  CAS  PubMed  Google Scholar 

  41. Ozaki T, Wu D, Sugimoto H, Nagase H, Nakagawara A (2013) Runt-related transcription factor 2 (RUNX2) inhibits p53-dependent apoptosis through the collaboration with HDAC6 in response to DNA damage (in eng). Cell Death Dis 4:e610. https://doi.org/10.1038/cddis.2013.127

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Wang H, Meng Y, Cui Q, Qin F, Yang H, Chen Y, Cheng Y, Shi J, Guo Y (2016) MiR-101 targets the EZH2/Wnt/beta-catenin the pathway to promote the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (in eng). Sci Rep 6:36988. https://doi.org/10.1038/srep36988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Hou Y, Li L, Ju Y, Lu Y, Chang L, Xiang X (2017) MiR-101-3p regulates the viability of lung squamous carcinoma cells via targeting EZH2 (in eng). J Cell Biochem 118:3142–3149. https://doi.org/10.1002/jcb.25836

    Article  CAS  PubMed  Google Scholar 

  44. Liu K, Jing Y, Zhang W, Fu X, Zhao H, Zhou X, Tao Y, Yang H, Zhang Y, Zen K, Zhang C, Li D, Shi Q (2017) Silencing miR-106b accelerates osteogenesis of mesenchymal stem cells and rescues against glucocorticoid-induced osteoporosis by targeting BMP2 (in eng). Bone 97:130–138. https://doi.org/10.1016/j.bone.2017.01.014

    Article  CAS  PubMed  Google Scholar 

  45. Zhang GP, Zhang J, Zhu CH, Lin L, Wang J, Zhang HJ, Li J, Yu XG, Zhao ZS, Dong W, Liu GB (2017) MicroRNA-98 regulates osteogenic differentiation of human bone mesenchymal stromal cells by targeting BMP2 (in eng). J Cell Mol Med 21:254–264. https://doi.org/10.1111/jcmm.12961

    Article  CAS  PubMed  Google Scholar 

  46. Sandbothe M, Buurman R, Reich N, Greiwe L, Vajen B, Gurlevik E, Schaffer V, Eilers M, Kuhnel F, Vaquero A, Longerich T, Roessler S, Schirmacher P, Manns MP, Illig T, Schlegelberger B, Skawran B (2017) The microRNA-449 family inhibits TGF-beta-mediated liver cancer cell migration by targeting SOX4 (in eng). J Hepatol 66:1012–1021. https://doi.org/10.1016/j.jhep.2017.01.004

    Article  CAS  PubMed  Google Scholar 

  47. Qiao L, Liu D, Li CG, Wang YJ (2018) MiR-203 is essential for the shift from osteogenic differentiation to adipogenic differentiation of mesenchymal stem cells in postmenopausal osteoporosis (in eng). Eur Rev Med Pharmacol Sci 22:5804–5814. https://doi.org/10.26355/eurrev_201809_15906

    Article  CAS  PubMed  Google Scholar 

  48. Kurihara H, Shinohara H, Yoshino H, Takeda K, Shiba H (2003) Neurotrophins in cultured cells from periodontal tissues (in eng). J Periodontol 74:76–84. https://doi.org/10.1902/jop.2003.74.1.76

    Article  CAS  PubMed  Google Scholar 

  49. Kendall HK, Haase HR, Li H, Xiao Y, Bartold PM (2000) Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts (in eng). J Periodontal Res 35:194–200

    Article  CAS  Google Scholar 

  50. Guo C, Li C, Yang K, Kang H, Xu X, Xu X, Deng L (2016) Increased EZH2 and decreased osteoblastogenesis during local irradiation-induced bone loss in rats (in eng). Sci Rep 6:31318. https://doi.org/10.1038/srep31318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

A portion of this work was carried out at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.

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Authors and Affiliations

  1. Department of Periodontal Medicine, Hiroshima University Graduate School of Biomedical and Health Sciences, Hiroshima, 734-8553, Japan

    Eri Kaneda-Ikeda, Tomoyuki Iwata, Noriyoshi Mizuno, Takayoshi Nagahara, Mikihito Kajiya, Kazuhisa Ouhara, Minami Yoshioka, Shu Ishida & Hidemi Kurihara

  2. Department of Department of General Dentistry, Hiroshima University Hospital, Hiroshima, 734-8553, Japan

    Hiroyuki Kawaguchi

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  1. Eri Kaneda-Ikeda
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  2. Tomoyuki Iwata
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  3. Noriyoshi Mizuno
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Contributions

EK-I: designed and performed the experiments; collected, analyzed, and interpreted the data; and wrote the manuscript; TI: designed the experiments with EK-I: analyzed the data, and edited the manuscript; NM, TN, MK, KO, MY, and SI: contributed to the experiments; HK: contributed to the experimental design; HK: supervised all the aspects of the study as the senior investigator and director of the laboratory.

Corresponding author

Correspondence to Tomoyuki Iwata.

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The authors declare no competing financial interests.

Ethical approval

This study was approved by the Ethics Committee of Hiroshima University Faculty of Dentistry (Hiroshima, Japan: approval no. E-D47-4).

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Informed consent was obtained from all HGF donor in this study.

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774_2019_1080_MOESM1_ESM.pdf

Fig. S1 IGF-1 induced various miRNAs and regulated markers of undifferentiated hMSCs or other osteogenic regulatory genes. hMSCs were cultured with IGF-1 (50 ng/mL) for 7 days (a), or hMSCs were transfected with the miR-101-3p mimic or the corresponding non-targeting miRNA. Cells were then cultured for 14 days in osteogenic induction medium with or without IGF-1 (50 ng/mL) (b). miR-101-3p expression on day 7 (a) and mRNAs for Smad7 and TWIST1 on day 14 (b) were determined with real-time PCR. The values represent miRNA expression levels normalized to U6 snRNA expression relative to the control and mRNA expression levels normalized to -actin mRNA expression relative to the control (Mean ± SD; **p < 0.01, *p < 0.05; Mann–Whitney U test; n = 4). (PDF 335 kb)

774_2019_1080_MOESM2_ESM.pdf

Fig. S2 miR-101-3p was changed in osteogenesis conditions and targeted Ezh2. hMSCs were cultured in growth medium or osteogenic induction medium for 14 days (a). hMSCs were transfected with the miR-101-3p mimic (b), miR-101-3p inhibitor (c), or the corresponding non-targeting miRNA, and then cultured for 14 days in osteogenic induction medium. miR-101-3p expression (a) and Ezh2 mRNA expression (b, c) were determined with real-time PCR. The values represent miRNA expression levels normalized to U6 snRNA expression relative to the control and mRNA expression levels normalized to -actin mRNA expression relative to the control (Mean ± SD; **p < 0.01, *p < 0.05; Mann–Whitney U test; n = 4). (PDF 536 kb)

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Kaneda-Ikeda, E., Iwata, T., Mizuno, N. et al. Regulation of osteogenesis via miR-101-3p in mesenchymal stem cells by human gingival fibroblasts. J Bone Miner Metab 38, 442–455 (2020). https://doi.org/10.1007/s00774-019-01080-2

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