微小RNA-451靶向調節鈣結合蛋白39對MSCs成骨分化的促進效應_《中國修復重建外科雜志》

作者：

康夏 ^1,2 , 康菲 ¹ , 楊波 ¹ , 郭洪峰 ¹ , 權毅 ² , 董世武 ¹

1第三軍醫大學解剖學教研室生物力學實驗室（重慶，400038）;;
2 解放軍成都軍區總醫院骨科;

關鍵詞：

微小RNA-451 鈣結合蛋白39 MSCs 成骨分化鈣沉積

DOI：

10.7507/1002-1892.20130245

視頻：

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目的探討微小RNA-451（micro RNA-451，miRNA-451）靶向調節鈣結合蛋白39（calcium binding protein 39，CAB39）表達，促進MSCs向成骨細胞分化的效應。方法選擇pMIR-report質粒及pRL-TK質粒，通過雙熒光素酶基因報告系統，鑒定CAB39是否為miRNA-451的靶基因。將pre-miRNA-451（A組）及anti-miRNA-451（C組）分別轉染到C3H10T1/2細胞中，同時設置相應的pre-miRNA陰性對照組（B組）和anti-miRNA陰性對照組（D組）；成骨誘導培養7、14 d，采用實時熒光定量PCR檢測成骨標志物Runx2、ALP mRNA相對表達量；誘導培養14 d，采用Western blot檢測細胞中CAB39蛋白表達，茜素紅染色觀察細胞外鈣基質沉積情況。結果經鑒定，CAB39是miRNA-451的靶基因。實時熒光定量PCR檢測示，成骨誘導培養7、14 d，A組Runx2、ALP mRNA相對表達量顯著高于B組，C組顯著低于D組，差異均有統計學意義（P lt; 0.05）。成骨誘導培養14 d，Western blot檢測示A組CAB39蛋白表達量為0.55 ± 0.05，較B組1.00 ± 0.07明顯降低；而C組為1.21 ± 0.05，較D組1.00 ± 0.04明顯增高；差異均有統計學意義（P lt; 0.05）。茜素紅染色示A組細胞外鈣基質沉積較B組明顯增多，而C組細胞外鈣基質沉積較D組明顯減少。結論miRNA-451可通過抑制CAB39表達，促進MSCs成骨分化。

引用本文： 康夏,康菲,楊波,郭洪峰,權毅,董世武. 微小RNA-451靶向調節鈣結合蛋白39對MSCs成骨分化的促進效應. 中國修復重建外科雜志, 2013, 27(9): 1122-1127. doi: 10.7507/1002-1892.20130245 復制

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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.
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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.
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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.

《中國修復重建外科雜志》

微小RNA-451靶向調節鈣結合蛋白39對MSCs成骨分化的促進效應

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《中國修復重建外科雜志》

微小RNA-451靶向調節鈣結合蛋白39對MSCs成骨分化的促進效應

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