Molecular Biology Reports

, Volume 43, Issue 3, pp 183–193 | Cite as

EGCG ameliorates the hypoxia-induced apoptosis and osteogenic differentiation reduction of mesenchymal stem cells via upregulating miR-210

  • Yiyan Qiu
  • Yang Chen
  • Tenghui Zeng
  • Weizhuang Guo
  • Wenyu Zhou
  • Xinjian YangEmail author
Original Article


The healing process of fractured bone is affected by the multiple factors regulating the growth and differentiation of osteoblasts and bone mesenchymal stem cells (MSCs), however, such markers and molecular events need to be orchestrated in details. This study investigated the effect of polyphenol(-)-epigallocatechin-3-gallate (EGCG) on the hypoxia-induced apoptosis and osteogenic differentiation of human bone marrow-derived MSCs, examined the miR-210 induction by EGCG, explored the target inhibition of the expression of receptor tyrosine kinase ligand ephrin-A3 (EFNA3) by miR-210, and then determined the association of the miR-210 promotion with the hypoxia-induced apoptosis and osteogenic differentiation. Results demonstrated that EGCG treatment significantly inhibited the hypoxia-induced apoptosis in MSCs and promoted the level of alkaline phosphatase (ALP), bone morphogenetic protein 2 (BMP-2), propeptide of type I procollagen I (PINP) and runt-related transcription factor 2 (RUNX2) in MSCs under either normoxia or hypoxia. Moreover, the EGCG treatment upregulated the miR-210 expression, in an association with EFNA3 downregulation; and the miR-210 upregulation significantly downregulated the expression of EFNA3 via the specific binding to the 3′ UTR of EFNA3. In addition, the manipulated miR-210 upregulation exerted amelioration on the hypoxia-induced apoptosis and on the hypoxia-reduced expression of ALP, BMP-2, PINP and RUNX2 in MSCs. In summary, our study indicated the protective role of EGCG in response to hypoxia and promontory role to osteogenic differentiation in MSCs via upregulating miR-210 and downregulating the expression of miR-210-targeted EFNA3. Our study implies the protective role of EGCG in the hypoxia-induced impairment in MSCs.


EGCG miR-210 Apoptosis Osteogenic differentiation Mesenchymal stem cells EFNA3 





Receptor tyrosine kinase ligand ephrin-A3


Mesenchymal stem cells


Alkaline phosphatase


Bone morphogenetic protein 2


Propeptide of type I procollagen I


Runt-related transcription factor 2


Hypoxia-inducible factors


Vascular endothelial growth factor






Protein tyrosine phosphatase-1B



Present study was supported by the Post-doctor Grant from the 2nd Shenzhen People’s Hospital (SZH2013006).

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interests regarding the publication of this article.


  1. 1.
    Louisia S, Stromboni M, Meunier A, Sedel L, Petite H (1999) Coral grafting supplemented with bone marrow. J Bone Joint Surg Br 81:719–724CrossRefPubMedGoogle Scholar
  2. 2.
    Harada S, Rodan GA (2003) Control of osteoblast function and regulation of bone mass. Nature 423:349–355CrossRefPubMedGoogle Scholar
  3. 3.
    Yao W, Lane NE (2015) Targeted delivery of mesenchymal stem cells to the bone. Bone 70:62–65PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Heppenstall RB, Goodwin CW, Goodwin CW, Brighton CT (1976) Fracture healing in the presence of chronic hypoxia. J Bone Joint Surg Am 58:1153–1156PubMedGoogle Scholar
  5. 5.
    Warren SM, Steinbrech DS, Mehrara BJ, Saadeh PB, Greenwald JA, Spector JA, Bouletreau PJ, Longaker MT (2001) Hypoxia regulates osteoblast gene expression. J Surg Res 99:147–155CrossRefPubMedGoogle Scholar
  6. 6.
    Akeno N, Czyzyk-Krzeska MF, Gross TS, Clemens TL (2001) Hypoxia induces vascular endothelial growth factor gene transcription in human osteoblast-like cells through the hypoxia-inducible factor-2alpha. Endocrinology 142:959–962PubMedGoogle Scholar
  7. 7.
    Bouletreau PJ, Warren SM, Spector JA, Peled ZM, Gerrets RP, Greenwald JA, Longaker MT (2002) Hypoxia and VEGF up-regulate BMP-2 mRNA and protein expression in microvascular endothelial cells: implications for fracture healing. Plast Reconstr Surg 109:2384–2397CrossRefPubMedGoogle Scholar
  8. 8.
    Wang HS, Han JS (2014) Research progress on combat trauma treatment in cold regions. Mil Med Res. 1:8PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Naik AA, Xie C, Zuscik MJ, Kingsley P, Schwarz EM, Awad H, Guldberg R, Drissi H, Puzas JE, Boyce B, Zhang X, O’Keefe RJ (2009) Reduced COX-2 expression in aged mice is associated with impaired fracture healing. J Bone Miner Res 24:251–264PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Gerstenfeld LC, Thiede M, Seibert K, Mielke C, Phippard D, Svagr B, Cullinane D, Einhorn TA (2003) Differential inhibition of fracture healing by non-selective and cyclooxygenase-2 selective non-steroidal anti-inflammatory drugs. J Orthop Res 21:670–675CrossRefPubMedGoogle Scholar
  11. 11.
    Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, O’Keefe RJ (2002) Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest 109:1405–1415PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Kolar P, Schmidt-Bleek K, Schell H, Gaber T, Toben D, Schmidmaier G, Perka C, Buttgereit F, Duda GN (2010) The early fracture hematoma and its potential role in fracture healing. Tissue Eng Part B 16:427–434CrossRefGoogle Scholar
  13. 13.
    Lu C, Saless N, Wang X, Sinha A, Decker S, Kazakia G, Hou H, Williams B, Swartz HM, Hunt TK, Miclau T, Marcucio RS (2013) The role of oxygen during fracture healing. Bone 52:220–229CrossRefPubMedGoogle Scholar
  14. 14.
    Scaringi R, Piccoli M, Papini N, Cirillo F, Conforti E, Bergante S, Tringali C, Garatti A, Gelfi C, Venerando B, Menicanti L, Tettamanti G, Anastasia L (2013) NEU3 sialidase is activated under hypoxia and protects skeletal muscle cells from apoptosis through the activation of the epidermal growth factor receptor signaling pathway and the hypoxia-inducible factor (HIF)-1alpha. J Biol Chem 288:3153–3162PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Piret JP, Mottet D, Raes M, Michiels C (2002) Is HIF-1alpha a pro- or an anti-apoptotic protein. Biochem Pharmacol 64:889–892CrossRefPubMedGoogle Scholar
  16. 16.
    Zhao X, Wang K, Hu F, Qian C, Guan H, Feng K, Zhou Y, Chen Z (2015) MicroRNA-101 protects cardiac fibroblasts from hypoxia-induced apoptosis via inhibition of the TGF-beta signaling pathway. Int J Biochem Cell Biol 65:155–164CrossRefPubMedGoogle Scholar
  17. 17.
    Ejtehadifar M, Shamsasenjan K, Movassaghpour A, Akbarzadehlaleh P, Dehdilani N, Abbasi P, Molaeipour Z, Saleh M (2015) The effect of hypoxia on mesenchymal stem cell biology. Adv Pharm Bull 5:141–149PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Sun G, Peng H (2015) HIF-1alpha-induced microRNA-210 reduces hypoxia-induced osteoblast MG-63 cell apoptosis. Biosci Biotechnol Biochem 79:1232–1239CrossRefPubMedGoogle Scholar
  19. 19.
    Kimura K, Nagano M, Salazar G, Yamashita T, Tsuboi I, Mishima H, Matsushita S, Sato F, Yamagata K, Ohneda O (2014) The role of CCL5 in the ability of adipose tissue-derived mesenchymal stem cells to support repair of ischemic regions. Stem Cells Dev 23:488–501PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Mountziaris PM, Dennis LE, Mountziaris I, Sing DC, Kasper FK, Mikos AG (2013) Effect of temporally patterned TNF-alpha delivery on in vitro osteogenic differentiation of mesenchymal stem cells cultured on biodegradable polymer scaffolds. J Biomater Sci Polym Ed 24:1794–1813PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Lee JS, Park JC, Kim TW, Jung BJ, Lee Y, Shim EK, Park S, Choi EY, Cho KS, Kim CS (2015) Human bone marrow stem cells cultured under hypoxic conditions present altered characteristics and enhanced in vivo tissue regeneration. Bone 78:34–45CrossRefPubMedGoogle Scholar
  22. 22.
    Hu N, Wang C, Liang X, Yin L, Luo X, Liu B, Zhang H, Shui W, Nan G, Wang N, Wu N, Chen X, He Y, Wen S, Deng F, Zhang H, Liao Z, Luu HH, Haydon RC, He TC, Huang W (2013) Inhibition of histone deacetylases potentiates BMP9-induced osteogenic signaling in mouse mesenchymal stem cells. Cell Physiol Biochem 32:486–498CrossRefPubMedGoogle Scholar
  23. 23.
    Gao Y, Li C, Wang H, Fan G (2015) Acceleration of bone-defect repair by using A-W MGC loaded with BMP2 and triple point-mutant HIF1alpha-expressing BMSCs. J Orthop Surg Res 10:83PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Huang J, Liu L, Feng M, An S, Zhou M, Li Z, Qi J, Shen H (2015) Effect of CoCl2 on fracture repair in a rat model of bone fracture. Mol Med Rep 12:5951–5956PubMedGoogle Scholar
  25. 25.
    Jiang C, Sun J, Dai Y, Cao P, Zhang L, Peng S, Zhou Y, Li G, Tang J, Xiang J (2015) HIF-1A and C/EBPs transcriptionally regulate adipogenic differentiation of bone marrow-derived MSCs in hypoxia. Stem Cell Res Ther 6:21PubMedCentralCrossRefPubMedGoogle Scholar
  26. 26.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233PubMedCentralCrossRefPubMedGoogle Scholar
  27. 27.
    Ambros V (2003) MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 113:673–676CrossRefPubMedGoogle Scholar
  28. 28.
    You L, Gu W, Chen L, Pan L, Chen J, Peng Y (2014) MiR-378 overexpression attenuates high glucose-suppressed osteogenic differentiation through targeting CASP3 and activating PI3 K/Akt signaling pathway. Int J Clin Exp Pathol 7:7249–7261PubMedCentralPubMedGoogle Scholar
  29. 29.
    Murata K, Ito H, Yoshitomi H, Yamamoto K, Fukuda A, Yoshikawa J, Furu M, Ishikawa M, Shibuya H, Matsuda S (2014) Inhibition of miR-92a enhances fracture healing via promoting angiogenesis in a model of stabilized fracture in young mice. J Bone Miner Res 29:316–326CrossRefPubMedGoogle Scholar
  30. 30.
    Jeon ES, Shin JH, Hwang SJ, Moon GJ, Bang OY, KiM HH (2014) Cobalt chloride induces neuronal differentiation of human mesenchymal stem cells through upregulation of microRNA-124a. Biochem Biophys Res Commun 444:581–587CrossRefPubMedGoogle Scholar
  31. 31.
    Liu XD, Cai F, Liu L, Zhang Y, Yang AL (2015) MicroRNA-210 is involved in the regulation of postmenopausal osteoporosis through promotion of VEGF expression and osteoblast differentiation. Biol Chem 396:339–347CrossRefPubMedGoogle Scholar
  32. 32.
    Xu J, Huang Z, Lin L, Fu M, Gao Y, Shen Y, Zou Y, Sun A, Qian J, Ge J (2014) miR-210 over-expression enhances mesenchymal stem cell survival in an oxidative stress environment through antioxidation and c-Met pathway activation. Sci China Life Sci 57:989–997CrossRefPubMedGoogle Scholar
  33. 33.
    Kim HW, Mallick F, Durrani S, Ashraf M, Jiang S, Haider KH (2012) Concomitant activation of miR-107/PDCD10 and hypoxamir-210/Casp8ap2 and their role in cytoprotection during ischemic preconditioning of stem cells. Antioxid Redox Signal 17:1053–1065PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Katiyar SK, Mukhtar H (1997) Tea antioxidants in cancer chemoprevention. J Cell Biochem Suppl 27:59–67CrossRefPubMedGoogle Scholar
  35. 35.
    Cao Y, Cao R (1999) Angiogenesis inhibited by drinking tea. Nature 398:381CrossRefPubMedGoogle Scholar
  36. 36.
    Chen CH, Kang L, Lin RW, Fu YC, Lin YS, Chang JK, Chen HT, Chen CH, Lin SY, Wang GJ, Ho ML (2013) (-)-Epigallocatechin-3-gallate improves bone microarchitecture in ovariectomized rats. Menopause 20:687–694CrossRefPubMedGoogle Scholar
  37. 37.
    Yagi H, Tan J, Tuan RS (2013) Polyphenols suppress hydrogen peroxide-induced oxidative stress in human bone-marrow derived mesenchymal stem cells. J Cell Biochem 114:1163–1173CrossRefPubMedGoogle Scholar
  38. 38.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  39. 39.
    Wang Z, Yin B, Wang B, Ma Z, Liu W, Lv G (2013) MicroRNA-210 promotes proliferation and invasion of peripheral nerve sheath tumor cells targeting EFNA3. Oncol Res 21:145–154CrossRefPubMedGoogle Scholar
  40. 40.
    Bandow K, Maeda A, Kakimoto K, Kusuyama J, Shamoto M, Ohnishi T, Matsuguchi T (2010) Molecular mechanisms of the inhibitory effect of lipopolysaccharide (LPS) on osteoblast differentiation. Biochem Biophys Res Commun 402:755–761CrossRefPubMedGoogle Scholar
  41. 41.
    Kamon M, Zhao R, Sakamoto K (2010) Green tea polyphenol (-)-epigallocatechin gallate suppressed the differentiation of murine osteoblastic MC3T3-E1 cells. Cell Biol Int 34:109–116Google Scholar
  42. 42.
    Nakagawa H, Wachi M, Woo JT, Kato M, Kasai S, Takahashi F, Lee IS, Nagai K (2002) Fenton reaction is primarily involved in a mechanism of (-)-epigallocatechin-3-gallate to induce osteoclastic cell death. Biochem Biophys Res Commun 292:94–101CrossRefPubMedGoogle Scholar
  43. 43.
    Wu CH, Yang YC, Yao WJ, Lu FH, Wu JS, Chang CJ (2002) Epidemiological evidence of increased bone mineral density in habitual tea drinkers. Arch Intern Med 162:1001–1006CrossRefPubMedGoogle Scholar
  44. 44.
    Vali B, Rao LG, El-Sohemy A (2007) Epigallocatechin-3-gallate increases the formation of mineralized bone nodules by human osteoblast-like cells. J Nutr Biochem 18:341–347CrossRefPubMedGoogle Scholar
  45. 45.
    Rodriguez R, Kondo H, Nyan M, Hao J, Miyahara T, Ohya K, Kasugai S (2011) Implantation of green tea catechin alpha-tricalcium phosphate combination enhances bone repair in rat skull defects. J Biomed Mater Res B 98:263–271CrossRefGoogle Scholar
  46. 46.
    Chen CH, Ho ML, Chang JK, Hung SH, Wang GJ (2005) Green tea catechin enhances osteogenesis in a bone marrow mesenchymal stem cell line. Osteoporos Int 16:2039–2045CrossRefPubMedGoogle Scholar
  47. 47.
    Jin P, Wu H, Xu G, Zheng L, Zhao J (2014) Epigallocatechin-3-gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic differentiation of mesenchymal stem cells from human bone marrow: an in vitro study. Cell Tissue Res 356:381–390CrossRefPubMedGoogle Scholar
  48. 48.
    Barile L, Lionetti V, Cervio E, Matteucci M, Gherghiceanu M, Popescu LM, Torre T, Siclari F, Moccetti T, Vassalli G (2014) Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction. Cardiovasc Res 103:530–541CrossRefPubMedGoogle Scholar
  49. 49.
    Ujigo S, Kamei N, Hadoush H, Fujioka Y, Miyaki S, Nakasa T, Tanaka N, Nakanishi K, Eguchi A, Sunagawa T, Ochi M (2014) Administration of microRNA-210 promotes spinal cord regeneration in mice. Spine 39:1099–1107CrossRefPubMedGoogle Scholar
  50. 50.
    Wang Z, Yin B, Wang B, Ma Z, Liu W, Lv G (2013) MicroRNA-210 promotes proliferation and invasion of peripheral nerve sheath tumor cells targeting EFNA3. Oncol Res 21:145–154CrossRefPubMedGoogle Scholar
  51. 51.
    Gomez-Maldonado L, Tiana M, Roche O, Prado-Cabrero A, Jensen L, Fernandez-Barral A, Guijarro-Munoz I, Favaro E, Moreno-Bueno G, Sanz L, Aragones J, Harris A, Volpert O, Jimenez B, Del PL (2015) EFNA3 long noncoding RNAs induced by hypoxia promote metastatic dissemination. Oncogene 34:2609–2620PubMedCentralCrossRefPubMedGoogle Scholar
  52. 52.
    Zhu R, Cho KS, Chen DF, Yang L (2014) Ephrin-A2 and -A3 are negative regulators of the regenerative potential of moller cells. Chin Med J (Engl 127:3438–3442Google Scholar
  53. 53.
    Pulkkinen K, Malm T, Turunen M, Koistinaho J, Yla-Herttuala S (2008) Hypoxia induces microRNA miR-210 in vitro and in vivo ephrin-A3 and neuronal pentraxin 1 are potentially regulated by miR-210. FEBS Lett 582:2397–2401CrossRefPubMedGoogle Scholar
  54. 54.
    Fasanaro P, D’Alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G, Capogrossi MC, Martelli F (2008) MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand ephrin-A3. J Biol Chem 283:15878–15883PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Yiyan Qiu
    • 1
  • Yang Chen
    • 1
  • Tenghui Zeng
    • 1
  • Weizhuang Guo
    • 1
  • Wenyu Zhou
    • 1
  • Xinjian Yang
    • 1
    Email author
  1. 1.Department of Spine SurgeryThe 2nd Shenzhen People’s HospitalShenzhenChina

Personalised recommendations