Effect of miR-21 down-regulated by H<sub>2</sub>O<sub>2</sub> on osteogenic differentiation of MC3T3-E1 cells _Chinese Journal of Reparative and Reconstructive Surgery

Authors：

PENG Jianqiang ¹ , HUANG Niansheng ¹ ^△ , HUANG Sheng ² , LI Liangping ³ , LING Zemin ³ , JIN Song ¹ , HUANG Aijun ¹ , LIN Kun ¹ ,  ZOU Xuenong ³

1. Department of Spine Surgery, the Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen Guangdong, 518033, P.R.China;
2. Department of Orthopedics, the First Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China;
3. Guangdong Provincial Key Laboratory of Orthopedics and Traumatology, Department of Spine Surgery, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou Guangdong, 510080, P.R.China;

Corresponding?author：

ZOU Xuenong, Email: zxnong@hotmail.com

Keywords：

H₂O₂; oxidative stress; miR-21; osteogenic differentiation; MC3T3-E1 cells

DOI：

10.7507/1002-1892.201707030

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ObjectiveTo explore the effect and mechanism of miR-21 down-regulated which was induced by H₂O₂ on osteogenic differentiation of MC3T3-E1 cells.MethodsMC3T3-E1 cells were cultured and passaged, and the 7th generation cells were harvested to use in experiment. The MC3T3-E1 cells were treated with different concentrations (0, 40, 80, 160, and 320 μmol/L) of H₂O₂. The expression of miR-21 was detected by real-time quantitative PCR (RT-PCR) and the cell viability was determined by MTS. Then the appropriate concentration of H₂O₂ was obtained. To analyze the effect of H₂O₂ on osteogenic differentiation of MC3T3-E1 cells, the MC3T3-E1 cells were divided into blank control group (group A), H₂O₂ group (group B), osteogenic induction group (group C), and H₂O₂+osteogenic induction group (group D). The expression of miR-21 and the osteogenesis related genes expressions of Runx2, osteopontin (OPN), and collagen type Ⅰ alpha 1 (Col1a1) were detected by RT-PCR. The expression of phosphatase and tensin homolog (PTEN) was detected by Western blot. The extracellular calcium deposition was detected by alizarin red staining. To analyze the effect on osteogenic differentiation of MC3T3-E1 cells after the transfection of miR-21 inhibitor and siRNA-PTEN, the MC3T3-E1 cells were divided into H₂O₂ group (group A1), H₂O₂+osteogenic induction group (group B1), H₂O₂+osteogenic induction+miR-21 inhibitor group (group C1), and H₂O₂+osteogenic induction+miR-21 inhibitor negative control group (group D1); and H₂O₂ group (group A2), H₂O₂+osteogenic induction group (group B2), H₂O₂+osteogenic induction+siRNA-PTEN negative control group (group C2), and H₂O₂+osteogenic induction+siRNA-PTEN group (group D2). The osteogenesis related genes were detected by RT-PCR and the extracellular calcium deposition was detected by alizarin red staining.ResultsThe results of MTS and RT-PCR showed that the appropriate concentration of H₂O₂ was 160 μmol/L. The expression of miR-21 was significantly lower in group B than in group A at 1 and 2 weeks (P<0.05). The expression of miR-21 was significantly lower in group D than in group C at 1 and 2 weeks (P<0.05). The expression of PTEN protein was significantly lower in group C than in groups A and D (P<0.05). The mRNA expressions of Runx2, OPN, and Col1a1 were significantly lower in group D than in group C at 1 and 2 weeks (P<0.05). The extracellular calcium deposition in group D was obviously less than that in group C. The expression of PTEN protein was significantly higher in group C1 than in group D1 (P<0.05). The mRNA expressions of Runx2 and OPN were significantly lower in group C1 than in groups B1 and D1 at 1 and 2 weeks (P<0.05). The mRNA expression of Col1a1 was significantly lower in group C1 than in groups B1 and D1 at 2 weeks (P<0.05). The extracellular calcium deposition in group C1 was obviously less than those in groups B1 and D1. The mRNA expressions of OPN and Col1a1 were significantly higher in group D2 than in groups B2 and C2 at 1 week (P<0.05). The extracellular calcium deposition in group D2 was obviously more than those in groups B2 and C2.ConclusionH₂O₂ inhibits the osteogenic differentiation of MC3T3-E1 cells, which may be induced by down-regulating the expression of miR-21.

Citation： PENG Jianqiang, HUANG Niansheng, HUANG Sheng, LI Liangping, LING Zemin, JIN Song, HUANG Aijun, LIN Kun, ZOU Xuenong. Effect of miR-21 down-regulated by H₂O₂ on osteogenic differentiation of MC3T3-E1 cells . Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(3): 276-284. doi: 10.7507/1002-1892.201707030 Copy

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2.	Zou X, Zou L, Foldager C, et al. Different mechanisms of spinal fusion using equine bone protein extract, rhBMP-2 and autograft during the process of anterior lumbar interbody fusion. Biomaterials, 2009, 30(6): 991-1004.
3.	Turgut A, G?ktürk E, K?se N, et al. Oxidant status increased during fracture healing in rats. Acta Orthop Scand, 1999, 70(5): 487-490.
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5.	Chen DF, Zhou ZY, Dai XJ, et al. Time-sequential changes of differentially expressed miRNAs during the process of anterior lumbar interbody fusion using equine bone protein extract, rhBMP-2 and autograft. Frontiers of Materials Science, 2014, 8(1): 72-86.
6.	Lv C, Hao YH, Han YX, et al. Role and mechanism of microRNA-21 in H₂O₂-induced apoptosis in bone marrow mesenchymal stem cells. J Clin Neurosci, 2016, 27: 154-160.
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8.	Lin Y, Liu X, Cheng Y, et al. Involvement of MicroRNAs in hydrogen peroxide-mediated gene regulation and cellular injury response in vascular smooth muscle cells. J Biol Chem, 2009, 284(12): 7903-7913.
9.	Meng YB, Li X, Li ZY, et al. microRNA-21 promotes osteogenic differentiation of mesenchymal stem cells by the PI3K/β-catenin pathway. J Orthop Res, 2015, 33(7): 957-964.
10.	Hays E, Schmidt J, Chandar N. Beta-catenin is not activated by downregulation of PTEN in osteoblasts. In Vitro Cell Dev Biol Anim, 2009, 45(7): 361-370.
11.	Manolagas SC. From estrogen-centric to aging and oxidative stress: a revised perspective of the pathogenesis of osteoporosis. Endocr Rev, 2010, 31(3): 266-300.
12.	Lin YC, Huang YC, Chen SC, et al. Neuroprotective effects of ugonin K on hydrogen peroxide-induced cell death in human neuroblastoma SH-SY5Y cells. Neurochem Res, 2009, 34(5): 923-930.
13.	Janssen-Heininger YM, Mossman BT, Heintz NH, et al. Redox-based regulation of signal transduction: principles, pitfalls, and promises. Free Radic Biol Med, 2008, 45(1): 1-17.
14.	Li H, Yang F, Wang Z, et al. MicroRNA-21 promotes osteogenic differentiation by targeting small mothers against decapentaplegic 7. Mol Med Rep, 2015, 12(1): 1561-1567.
15.	Sánchez-Rodríguez MA, Ruiz-Ramos M, Correa-Mu?oz E, et al. Oxidative stress as a risk factor for osteoporosis in elderly Mexicans as characterized by antioxidant enzymes. BMC Musculoskelet Disord, 2007, 8: 124.
16.	Wauquier F, Leotoing L, Coxam V, et al. Oxidative stress in bone remodelling and disease. Trends Mol Med, 2009, 15(10): 468-477.
17.	Wang F, Yin P, Lu Y, et al. Cordycepin prevents oxidative stress-induced inhibition of osteogenesis. Oncotarget, 2015, 6(34): 35496-35508.
18.	Lee D, Kook SH, Ji H, et al. N-acetyl cysteine inhibits H₂O₂-mediated reduction in the mineralization of MC3T3-E1 cells by down-regulating Nrf2/HO-1 pathway. BMB Rep, 2015, 48(11): 636-641.
19.	Gericke A, Munson M, Ross AH. Regulation of the PTEN phosphatase. Gene, 2006, 374: 1-9.
20.	Burgers TA, Hoffmann MF, Collins CJ, et al. Mice lacking pten in osteoblasts have improved intramembranous and late endochondral fracture healing. PLoS One, 2013, 8(5): e63857.
21.	Guntur AR, Reinhold MI, Cuellar JJ, et al. Conditional ablation of Pten in osteoprogenitors stimulates FGF signaling. Development, 2011, 138(7): 1433-1444.
22.	Yang P, Peairs JJ, Tano R, et al. Oxidant-mediated Akt activation in human RPE cells. Invest Ophthalmol Vis Sci, 2006, 47(10): 4598-4606.
23.	Yan-nan B, Zhao-yan Y, Li-xi L, et al. MicroRNA-21 accelerates hepatocyte proliferation in vitro via PI3K/Akt signaling by targeting PTEN. Biochem Biophys Res Commun, 2014, 443(3): 802-807.
24.	Wei J, Feng L, Li Z, et al. MicroRNA-21 activates hepatic stellate cells via PTEN/Akt signaling. Biomed Pharmacother, 2013, 67(5): 387-392.
25.	Almeida M. Unraveling the role of FoxOs in bone-insights from mouse models. Bone, 2011, 49(3): 319-327.
26.	Kousteni S. FoxOs: Unifying links between oxidative stress and skeletal homeostasis. Curr Osteoporos Rep, 2011, 9(2): 60-66.
27.	Ambrogini E, Almeida M, Martin-Millan M, et al. FoxO-mediated defense against oxidative stress in osteoblasts is indispensable for skeletal homeostasis in mice. Cell Metab, 2010, 11(2): 136-146.
28.	Wang K, Li PF. Foxo3a regulates apoptosis by negatively targeting miR-21. J Biol Chem, 2010, 285(22): 16958-16966.

1. Ai-Aql ZS, Alagl AS, Graves DT, et al. Molecular mechanisms controlling bone formation during fracture healing and distraction osteogenesis. J Dent Res, 2008, 87(2): 107-118.
2. Zou X, Zou L, Foldager C, et al. Different mechanisms of spinal fusion using equine bone protein extract, rhBMP-2 and autograft during the process of anterior lumbar interbody fusion. Biomaterials, 2009, 30(6): 991-1004.
3. Turgut A, G?ktürk E, K?se N, et al. Oxidant status increased during fracture healing in rats. Acta Orthop Scand, 1999, 70(5): 487-490.
4. Jebahi S, Nsiri R, Boujbiha M, et al. The impact of orthopedic device associated with carbonated hydroxyapatite on the oxidative balance: experimental study of bone healing rabbit model. Eur J Orthop Surg Traumatol, 2013, 23(7): 759-766.
5. Chen DF, Zhou ZY, Dai XJ, et al. Time-sequential changes of differentially expressed miRNAs during the process of anterior lumbar interbody fusion using equine bone protein extract, rhBMP-2 and autograft. Frontiers of Materials Science, 2014, 8(1): 72-86.
6. Lv C, Hao YH, Han YX, et al. Role and mechanism of microRNA-21 in H₂O₂-induced apoptosis in bone marrow mesenchymal stem cells. J Clin Neurosci, 2016, 27: 154-160.
7. Cheng Y, Liu X, Zhang S, et al. MicroRNA-21 protects against the H(2)O(2)-induced injury on cardiac myocytes via its target gene PDCD4. J Mol Cell Cardiol, 2009, 47(1): 5-14.
8. Lin Y, Liu X, Cheng Y, et al. Involvement of MicroRNAs in hydrogen peroxide-mediated gene regulation and cellular injury response in vascular smooth muscle cells. J Biol Chem, 2009, 284(12): 7903-7913.
9. Meng YB, Li X, Li ZY, et al. microRNA-21 promotes osteogenic differentiation of mesenchymal stem cells by the PI3K/β-catenin pathway. J Orthop Res, 2015, 33(7): 957-964.
10. Hays E, Schmidt J, Chandar N. Beta-catenin is not activated by downregulation of PTEN in osteoblasts. In Vitro Cell Dev Biol Anim, 2009, 45(7): 361-370.
11. Manolagas SC. From estrogen-centric to aging and oxidative stress: a revised perspective of the pathogenesis of osteoporosis. Endocr Rev, 2010, 31(3): 266-300.
12. Lin YC, Huang YC, Chen SC, et al. Neuroprotective effects of ugonin K on hydrogen peroxide-induced cell death in human neuroblastoma SH-SY5Y cells. Neurochem Res, 2009, 34(5): 923-930.
13. Janssen-Heininger YM, Mossman BT, Heintz NH, et al. Redox-based regulation of signal transduction: principles, pitfalls, and promises. Free Radic Biol Med, 2008, 45(1): 1-17.
14. Li H, Yang F, Wang Z, et al. MicroRNA-21 promotes osteogenic differentiation by targeting small mothers against decapentaplegic 7. Mol Med Rep, 2015, 12(1): 1561-1567.
15. Sánchez-Rodríguez MA, Ruiz-Ramos M, Correa-Mu?oz E, et al. Oxidative stress as a risk factor for osteoporosis in elderly Mexicans as characterized by antioxidant enzymes. BMC Musculoskelet Disord, 2007, 8: 124.
16. Wauquier F, Leotoing L, Coxam V, et al. Oxidative stress in bone remodelling and disease. Trends Mol Med, 2009, 15(10): 468-477.
17. Wang F, Yin P, Lu Y, et al. Cordycepin prevents oxidative stress-induced inhibition of osteogenesis. Oncotarget, 2015, 6(34): 35496-35508.
18. Lee D, Kook SH, Ji H, et al. N-acetyl cysteine inhibits H₂O₂-mediated reduction in the mineralization of MC3T3-E1 cells by down-regulating Nrf2/HO-1 pathway. BMB Rep, 2015, 48(11): 636-641.
19. Gericke A, Munson M, Ross AH. Regulation of the PTEN phosphatase. Gene, 2006, 374: 1-9.
20. Burgers TA, Hoffmann MF, Collins CJ, et al. Mice lacking pten in osteoblasts have improved intramembranous and late endochondral fracture healing. PLoS One, 2013, 8(5): e63857.
21. Guntur AR, Reinhold MI, Cuellar JJ, et al. Conditional ablation of Pten in osteoprogenitors stimulates FGF signaling. Development, 2011, 138(7): 1433-1444.
22. Yang P, Peairs JJ, Tano R, et al. Oxidant-mediated Akt activation in human RPE cells. Invest Ophthalmol Vis Sci, 2006, 47(10): 4598-4606.
23. Yan-nan B, Zhao-yan Y, Li-xi L, et al. MicroRNA-21 accelerates hepatocyte proliferation in vitro via PI3K/Akt signaling by targeting PTEN. Biochem Biophys Res Commun, 2014, 443(3): 802-807.
24. Wei J, Feng L, Li Z, et al. MicroRNA-21 activates hepatic stellate cells via PTEN/Akt signaling. Biomed Pharmacother, 2013, 67(5): 387-392.
25. Almeida M. Unraveling the role of FoxOs in bone-insights from mouse models. Bone, 2011, 49(3): 319-327.
26. Kousteni S. FoxOs: Unifying links between oxidative stress and skeletal homeostasis. Curr Osteoporos Rep, 2011, 9(2): 60-66.
27. Ambrogini E, Almeida M, Martin-Millan M, et al. FoxO-mediated defense against oxidative stress in osteoblasts is indispensable for skeletal homeostasis in mice. Cell Metab, 2010, 11(2): 136-146.
28. Wang K, Li PF. Foxo3a regulates apoptosis by negatively targeting miR-21. J Biol Chem, 2010, 285(22): 16958-16966.

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Effect of miR-21 down-regulated by H₂O₂ on osteogenic differentiation of MC3T3-E1 cells

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Effect of miR-21 down-regulated by H₂O₂ on osteogenic differentiation of MC3T3-E1 cells