MICRO RNA-451 PROMOTING OSTEOGENESIS OF MESENCHYMAL STEM CELLS BY TARGETING REGULATORY CALCIUM BINDING PROTEIN 39_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

KANG Xia ^1,2 , KANG Fei ¹ , YANG Bo ¹ , GUO Hongfeng ¹ , QUAN Yi ² , DONG Shiwu ¹

1Laboratory of Biomechanics, Department of Anatomy, the Third Military Medical University, Chongqing, 400038, P.R.China;;
2Department of Orthopaedics, General Hospital of Chengdu Military Region of Chinese PLA. Corresponding author: QUAN Yi, E-mail: quanyi56@sina.com;;
DONG Shiwu, E-mail: dongshiwu@163.com;

Corresponding author：

Keywords：

Micro RNA-451; Calcium binding protein 39; Mesenchymal stem cells; Osteogenesis; Calcium deposition

DOI：

10.7507/1002-1892.20130245

Video：

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Objective To investigate the role of micro RNA-451 (miRNA-451) in promoting the osteogenesis of mesenchymal stem cells (MSCs) by targeting regulatory calcium binding protein 39 (CAB39). Methods pMIR-report and pRL-TK vectors were selected to identify the relationship between miRNA-451 and CAB39 by using dual-luciferase reporter assay. pre-miRNA-451 (group A), anti-miRNA-451 (group C), pre-miRNA negative control (group B), and anti-miRNA negative control (group D) were transfected into the C3H10T1/2 cells, respectively. Then, the cells were collected after osteogenic induction for 7 and 14 days. At 7 and 14 days, the real-time fluorescent quantitative PCR and Western blot assays were performed to detect the related osteogenetic biomarkers [Runx2 and alkaline phosphatase (ALP) mRNA] and expressions of CAB39 protein. At 14 days, the extracellular calcium deposition during the osteogenesis of MSCs was tested by Alizarin red staining method. Results CAB39 was the target gene of miRNA-451. At 7 and 14 days after osteogenic induction, the mRNA expressions of Runx2 and ALP in group A were significantly higher than those in group B (P lt; 0.05), and the expressions in group C was significantly lower than those in group D (P lt; 0.05). Furthermore, at 14 days after osteogenic induction, the protein expression of CAB39 in group A (0.55 ± 0.05) was significantly lower than that in group B (1.00 ± 0.07), and the protein expression in group C (1.21 ± 0.05) was significantly higher than that in group D (1.00 ± 0.04), all showing significant difference (P lt; 0.05). Finally, at 14 days after osteogenic induction, the extracellular calcium deposition in group A was obviously more than that in group B, and group C was downregulated when compared with group D. Conclusion miRNA-451 can promote the osteogenesis process of MSCs by downregulating the CAB39.

Citation： KANG Xia,KANG Fei,YANG Bo,GUO Hongfeng,QUAN Yi,DONG Shiwu. MICRO RNA-451 PROMOTING OSTEOGENESIS OF MESENCHYMAL STEM CELLS BY TARGETING REGULATORY CALCIUM BINDING PROTEIN 39. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(9): 1122-1127. doi: 10.7507/1002-1892.20130245 Copy

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15.	Goldoni D, Yarham JM, McGahon MK, et al. A novel dual-fluorescence strategy for functionally validating microRNA targets in 3’ untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212. Biochem J, 2012, 448(1): 103-113.
16.	Yang B, Guo H, Zhang Y, et al. MicroRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9. PLoS One, 2011, 6(7): e21679.
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18.	Mizutani J, Tokuda H, Matsushima-Nishiwaki R, et al. Involvement of AMP-activated protein kinase in TGF-β-stimulated VEGF synthesis inosteoblasts. Int J Mol Med, 2012, 29(4): 550-556.
19.	Tian Y, Nan Y, Han L, et al. MicroRNA miR-451 downregulates the PI3K/AKT pathway through CAB39 in human glioma. Int J Oncol, 2012, 40(4): 1105-1112.
20.	Wang X, Zhu H, Zhang X, et al. Loss of the miR-144/451 cluster impairs ischaemic preconditioning-mediated cardioprotection by targeting Rac-1. Cardiovasc Res, 2012, 94(2): 379-390.
21.	Redova M, Poprach A, Nekvindova J, et al. Circulating miR-378 and miR-451 in serum are potential biomarkers for renal cell carcinoma. J Transl Med, 2012, 10: 55.
22.	Tanaka-Watanabe Y, Asahara H. Joint and microRNA. Nihon Rinsho Meneki Gakkai Kaishi, 2012, 35(6): 447-454.
23.	Ouyang L, Liu P, Yang S, et al. A three-plasma miRNA signature serves as novel biomarkers for osteosarcoma. Med Oncol, 2013, 30(1): 340.
24.	Hu H, Zhang Y, Cai XH, et al. Changes in microRNA expression in the MG-63 osteosarcoma cell line compared with osteoblasts. Oncol Lett, 2012, 4(5): 1037-1042.
25.	Hassan MQ, Maeda Y, Taipaleenmaki H, et al. miR-218 directs a Wnt signaling circuit to promote differentiation of osteoblasts and osteomimicry of metastatic cancer cells. J Biol Chem, 2012, 287(50): 42084-42092.

1. Boudeau J, Baas AF, Deak M, et al. MO25 alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm. EMBO J, 2003, 22(19): 5102-5114.
2. Brajenovic M, Joberty G, Küster B, et al. Comprehensive proteomic analysis of human Par protein complexes reveals an interonnected protein network. J Biol Chem, 2004, 279(13): 12804-12811.
3. Jeyabalan J, Shah M, Viollet B, et al. AMP-activated protein kinase pathway and bone metabolism. J Endocrinol, 2012, 212(3): 277-290.
4. Bakhshandeh B, Hafizi M, Ghaemi N, et al. Down-regulation of miRNA-221 triggers osteogenic differentiation in human stem cells. Biotechnol Lett, 2012, 34(8): 1579-1587.
5. Kim KM, Park SJ, Jung SH, et al. miR-182 is a negative regulator of osteoblast proliferation, differentiation, and skeletogenesis through targeting FoxO1. J Bone Miner Res, 2012, 27(8): 1669-1679.
6. Laine SK, Alm JJ, Virtanen SP, et al. MicroRNAs miR-96, miR-124, and miR-199a regulate gene expression in human bone marrow-derived mesenchymal stem cells. J Cell Biochem, 2012, 113(8): 2687-2695.
7. Chen Q, Liu W, Sinha KM, et al. Identification and characterization of MicroRNAs controlled by the osteoblast-specific transcription factor osterix. PLoS One, 2013, 8(3): e58104.
8. Hawley SA, Boudeau J, Reid JL, et al. Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol, 2003, 2(4): 28.
9. Woods SC, Gotoh K, Clegg DJ. Gender differences in the control of energy homeostasis. Exp Biol Med (Maywood), 2003, 228(10): 1175-1180.
10. Utting JC, Robins SP, Brandao-Burch A, et al. Hypoxia inhibits the growth, differentiation and bone-forming capacity of rat osteoblasts. Exp Cell Res, 2006, 312(10): 1693-1702.
11. Lee WH, Kim SG. AMPK-dependent metabolic regulation by PPAR agonists. PPAR Research, 2010, (2010): 549101.
12. Shah M, Kola B, Bataveljic A, et al. AMP-activated protein kinase (AMPK) activation regulates in vitro bone formation and bone mass. Bone, 2010, 47(2): 309-319.
13. Kasai T, Bandow K, Suzuki H, et al. Osteoblast differentiation is functionally associated with decreased AMP kinase activity. J Cell Physiol, 2009, 221(3): 740-749.
14. Jeyabalan J, Shah M, Viollet B, et al. Mice lacking AMP-activated protein kinase α1 catalytic subunit have increased bone remodelling and modified skeletal responses to hormonal challenges induced by ovariectomy and intermittent PTH treatment. J Endocrinol, 2012, 214(3): 349-358.
15. Goldoni D, Yarham JM, McGahon MK, et al. A novel dual-fluorescence strategy for functionally validating microRNA targets in 3’ untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212. Biochem J, 2012, 448(1): 103-113.
16. Yang B, Guo H, Zhang Y, et al. MicroRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9. PLoS One, 2011, 6(7): e21679.
17. Wang SS, Huang HY, Chen SZ, et al. Gdf6 induces commitment of pluripotent mesenchymal C3H10T1/2 cells to the adipocyte lineage. FEBS J, 2013. [Epub ahead of print].
18. Mizutani J, Tokuda H, Matsushima-Nishiwaki R, et al. Involvement of AMP-activated protein kinase in TGF-β-stimulated VEGF synthesis inosteoblasts. Int J Mol Med, 2012, 29(4): 550-556.
19. Tian Y, Nan Y, Han L, et al. MicroRNA miR-451 downregulates the PI3K/AKT pathway through CAB39 in human glioma. Int J Oncol, 2012, 40(4): 1105-1112.
20. Wang X, Zhu H, Zhang X, et al. Loss of the miR-144/451 cluster impairs ischaemic preconditioning-mediated cardioprotection by targeting Rac-1. Cardiovasc Res, 2012, 94(2): 379-390.
21. Redova M, Poprach A, Nekvindova J, et al. Circulating miR-378 and miR-451 in serum are potential biomarkers for renal cell carcinoma. J Transl Med, 2012, 10: 55.
22. Tanaka-Watanabe Y, Asahara H. Joint and microRNA. Nihon Rinsho Meneki Gakkai Kaishi, 2012, 35(6): 447-454.
23. Ouyang L, Liu P, Yang S, et al. A three-plasma miRNA signature serves as novel biomarkers for osteosarcoma. Med Oncol, 2013, 30(1): 340.
24. Hu H, Zhang Y, Cai XH, et al. Changes in microRNA expression in the MG-63 osteosarcoma cell line compared with osteoblasts. Oncol Lett, 2012, 4(5): 1037-1042.
25. Hassan MQ, Maeda Y, Taipaleenmaki H, et al. miR-218 directs a Wnt signaling circuit to promote differentiation of osteoblasts and osteomimicry of metastatic cancer cells. J Biol Chem, 2012, 287(50): 42084-42092.

Chinese Journal of Reparative and Reconstructive Surgery

MICRO RNA-451 PROMOTING OSTEOGENESIS OF MESENCHYMAL STEM CELLS BY TARGETING REGULATORY CALCIUM BINDING PROTEIN 39

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