CHANGES OF MICRO RNA AND TARGET GENE EXPRESSION LEVELS IN OSTEOGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WEI Junqiang ¹ , ZHANG Boxun ¹ , ZHENG Xiaofei ² , CHEN Hua ¹ , TIAN Xuezhong ¹ , TANG Peifu ¹ , SONG Qing ³ , LI Tanshi. ³

1Department of Orthopaedics, 3Department of Emergency,Chinese PLA General Hospital, Beijing, 100853, P.R.China;;
2Institute of Radiation Medicine, Academy of Military Medical Sciences.;

Corresponding author：

Keywords：

Micro RNA; Target gene; Human bone marrow mesenchymal stem cells; Osteogenic differentiation

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Objective To clarify the trends of expression levels of several up-regulated micro RNA (miRNA) in tissues of atrophic bone nonunion and mRNAs and proteins of their related target genes in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), and to explore their biological functions. Methods The hBMSCs were isolated from bone marrow of il iac bone by gradient centrifugation, and cultured. Osteogenic culture medium was used for osteogenic differentiation of the 4th generation of hBMSCs. The changes of corresponding miRNAs, mRNA and protein expression levels of related target genes were observed at 0 hour, 12 hours, 1 day, 2 days, 4 days, 7 days, and 14 days, by quantitative real-time PCR and Western blot. Results In the process of hBMSCs osteogenic differentiation, the mRNA and protein expression levels of osteoblastic target genes [alkal ine phosphatase l iver/bone/kidney (ALPL), bone morphogenetic
protein 2 (BMP-2), and platelet-derived factor alpha polypeptide (PDGF-A)] at most time points increased significantly when
compared with the values at 0 hour except that of BMP-2 decreased at 12 hours and 1 day, with maximum changes at 1 to 7 days. The miRNA expression levels, mRNA and protein expression levels changed significantly at different time points, while the trends of hsa-miRNA-149 and hsa-miRNA-654-5p changes were negatively correlated with the trends of ALPL and BMP-2 mRNA and protein expression changes respectively (P lt; 0.05). There was no obviously negative correlation between the trends of hsa-miRNA-221 change and PDGF-A change (P gt; 0.05). Conclusion In the osteogenic differentiation process of hBMSCs, hsa-miRNA-149 and hsa-miRNA-654-5p are closely related with the mRNA and protein regulation of ALPL and BMP-2, respectively.

Citation： WEI Junqiang,ZHANG Boxun,ZHENG Xiaofei,CHEN Hua,TIAN Xuezhong,TANG Peifu,SONG Qing,LI Tanshi.. CHANGES OF MICRO RNA AND TARGET GENE EXPRESSION LEVELS IN OSTEOGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(4): 483-487. doi: Copy

1. 魏均强. miRNA在萎缩性骨不连中的调控作用及其分子生物学机制. 北京: 中国人民解放军军医进修学院, 2011.

2. Service RF. Tissue engineers build new bone. Science, 2000, 289(5484): 1498-1500.

3. Satija NK, Gurudutta GU, Sharma S, et al. Mesenchymal stem cells: molecular targets for tissue engineering. Stem Cells Dev, 2007, 16(1): 7-23.

4. Xie C, Reynolds D, Awad H, et al. Structural bone allograft combined with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering. Tissue Eng, 2007, 13(3): 435-445.

5. Tang Y, Tang W, Lin Y, et al. Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats. Cell Biol Int, 2008, 32(9): 1150-1157.

6. Kawabata K, Sakurai F, Koizumi N, et al. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm, 2006, 3(2): 95-103.

7. 胡炜, 俞兴, 徐林. 骨髓基质细胞诱导分化成骨方法及相关研究进展. 中国组织工程研究与临床康复, 2009, 13(1): 169-172.

8. Niemeyer P, Krause U, Punzel M, et al. Mesenchymal stem cells for tissue engineering of bone: 3D-cultivation and osteogenic differentiation on mineralized collagen. Z Orthop Ihre Grenzgeb, 2003, 141(6): 712-717.

9. Taira M, Nakao H, Takahashi J, et al. Effects of two vitamins, two growth factors and dexamethasone on the proliferation of rat bone marrow stromal cells and osteoblastic MC3T3-E1cells. J Oral Rehabil, 2003, 30(7): 697-701.

10. Rodríguez JP, González M, Ríos S, et al. Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J Cell Biochem, 2004, 93(4): 721-731.

11. Otsuka E, Yamaguchi A, Shigehisa H, et al. Characterizations of osteoblastic differentiation of stromalcell line ST2 that is induced by ascorbic acid. Am J Physiol, 1999, 277(1 Pt 1): C132-138.

12. Egusa H, Iida K, Kobayashi M, et al. Downregulation of extracellular matrix-related gene clusters during osteogenic differentiation of human bone marrow- and adipose tissue-derived stromal cells. Tissue Eng, 2007, 13(10): 2589-2600.

13. Timmer NM, van Horssen J, Otte-Holler I, et al. Amyloid beta induces cellular relocalization and production of agrin and glypican-1. Brain Res, 2009, 1260: 38-46.

14. Oreffo RO, Cooper C, Mason C, et al. Mesenchymal stem cells: lineage, plasticity, and skeletal therapeutic potential. Stem Cell Rev, 2005, 1(2): 169-178.

15. Kimelman N, Pelled G, Gazit Z, et al. Applications of gene therapy and adult stem cells in bone bioengineering. Regen Med, 2006, 1(4): 549-561.

16. Gugala Z, Olmsted-Davis EA, Gannon FH, et al. Osteo-induction by ex vivo adenovirus-mediated BMP-2 delivery is independent of cell type. Gene Ther, 2003, 10(16): 1289-1296.

17. Gugala Z, Davis AR, Fouletier-Dilling CM, et al. Adenovirus BMP-2 induced osteogenesis in combination with collagen carriers. Biomaterials, 2007, 28(30): 4469-4479.

18. Sipos W, Pietschmann P, Rauner M. Strategies for novel therapeutic approaches targeting cytokines and signaling pathways of osteoclasto- and osteoblastogenesis in the fight against immune-mediated bone and joint diseases. Curr Med Chem, 2008, 15(2): 127-136.

19. Alcaraz MJ, Megias J, Garcia-Arnandis I, et al. New molecular targets for the treatment of osteoarthritis. Biochem Pharmacol, 2010, 80(1): 13-21.

20. Fulci V, Scappucci G, Sebastiani GD, et al. miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis. Hum Immunol, 2010, 71(2): 206-211.

21. Luo X, Tsai LM, Shen N, et al. Evidence for microRNA-mediated regulation in rheumatic diseases. Ann Rheum Dis, 2010, 69 Suppl 1: i30-36.

22. Miyaki S, Sato T, Inoue A, et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev, 2010, 24(11): 1173-1185.

23. Babkina IV, Osipov DA, Solovyov YN, et al. Endostatin, placental growth factor, and fibroblast growth factors-1 and -2 in the sera of patients with primary osteosarcomas. Bull Exp Biol Med, 2009, 148(2): 246-249.

1. 魏均强. miRNA在萎缩性骨不连中的调控作用及其分子生物学机制. 北京: 中国人民解放军军医进修学院, 2011.

2. Service RF. Tissue engineers build new bone. Science, 2000, 289(5484): 1498-1500.

3. Satija NK, Gurudutta GU, Sharma S, et al. Mesenchymal stem cells: molecular targets for tissue engineering. Stem Cells Dev, 2007, 16(1): 7-23.

4. Xie C, Reynolds D, Awad H, et al. Structural bone allograft combined with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering. Tissue Eng, 2007, 13(3): 435-445.

5. Tang Y, Tang W, Lin Y, et al. Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats. Cell Biol Int, 2008, 32(9): 1150-1157.

6. Kawabata K, Sakurai F, Koizumi N, et al. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm, 2006, 3(2): 95-103.

7. 胡炜, 俞兴, 徐林. 骨髓基质细胞诱导分化成骨方法及相关研究进展. 中国组织工程研究与临床康复, 2009, 13(1): 169-172.

8. Niemeyer P, Krause U, Punzel M, et al. Mesenchymal stem cells for tissue engineering of bone: 3D-cultivation and osteogenic differentiation on mineralized collagen. Z Orthop Ihre Grenzgeb, 2003, 141(6): 712-717.

9. Taira M, Nakao H, Takahashi J, et al. Effects of two vitamins, two growth factors and dexamethasone on the proliferation of rat bone marrow stromal cells and osteoblastic MC3T3-E1cells. J Oral Rehabil, 2003, 30(7): 697-701.

10. Rodríguez JP, González M, Ríos S, et al. Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J Cell Biochem, 2004, 93(4): 721-731.

11. Otsuka E, Yamaguchi A, Shigehisa H, et al. Characterizations of osteoblastic differentiation of stromalcell line ST2 that is induced by ascorbic acid. Am J Physiol, 1999, 277(1 Pt 1): C132-138.

12. Egusa H, Iida K, Kobayashi M, et al. Downregulation of extracellular matrix-related gene clusters during osteogenic differentiation of human bone marrow- and adipose tissue-derived stromal cells. Tissue Eng, 2007, 13(10): 2589-2600.

13. Timmer NM, van Horssen J, Otte-Holler I, et al. Amyloid beta induces cellular relocalization and production of agrin and glypican-1. Brain Res, 2009, 1260: 38-46.

14. Oreffo RO, Cooper C, Mason C, et al. Mesenchymal stem cells: lineage, plasticity, and skeletal therapeutic potential. Stem Cell Rev, 2005, 1(2): 169-178.

15. Kimelman N, Pelled G, Gazit Z, et al. Applications of gene therapy and adult stem cells in bone bioengineering. Regen Med, 2006, 1(4): 549-561.

16. Gugala Z, Olmsted-Davis EA, Gannon FH, et al. Osteo-induction by ex vivo adenovirus-mediated BMP-2 delivery is independent of cell type. Gene Ther, 2003, 10(16): 1289-1296.

17. Gugala Z, Davis AR, Fouletier-Dilling CM, et al. Adenovirus BMP-2 induced osteogenesis in combination with collagen carriers. Biomaterials, 2007, 28(30): 4469-4479.

18. Sipos W, Pietschmann P, Rauner M. Strategies for novel therapeutic approaches targeting cytokines and signaling pathways of osteoclasto- and osteoblastogenesis in the fight against immune-mediated bone and joint diseases. Curr Med Chem, 2008, 15(2): 127-136.

19. Alcaraz MJ, Megias J, Garcia-Arnandis I, et al. New molecular targets for the treatment of osteoarthritis. Biochem Pharmacol, 2010, 80(1): 13-21.

20. Fulci V, Scappucci G, Sebastiani GD, et al. miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis. Hum Immunol, 2010, 71(2): 206-211.

21. Luo X, Tsai LM, Shen N, et al. Evidence for microRNA-mediated regulation in rheumatic diseases. Ann Rheum Dis, 2010, 69 Suppl 1: i30-36.

22. Miyaki S, Sato T, Inoue A, et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev, 2010, 24(11): 1173-1185.

23. Babkina IV, Osipov DA, Solovyov YN, et al. Endostatin, placental growth factor, and fibroblast growth factors-1 and -2 in the sera of patients with primary osteosarcomas. Bull Exp Biol Med, 2009, 148(2): 246-249.

Chinese Journal of Reparative and Reconstructive Surgery

CHANGES OF MICRO RNA AND TARGET GENE EXPRESSION LEVELS IN OSTEOGENIC DIFFERENTIATION OF HUMAN BONE MARROW MESENCHYMAL STEM CELLS

Abstract Full text Figures/Tables Video References Cited by

Format

Content

1.	魏均强. miRNA在萎缩性骨不连中的调控作用及其分子生物学机制. 北京: 中国人民解放军军医进修学院, 2011.
2.	Service RF. Tissue engineers build new bone. Science, 2000, 289(5484): 1498-1500.
3.	Satija NK, Gurudutta GU, Sharma S, et al. Mesenchymal stem cells: molecular targets for tissue engineering. Stem Cells Dev, 2007, 16(1): 7-23.
4.	Xie C, Reynolds D, Awad H, et al. Structural bone allograft combined with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering. Tissue Eng, 2007, 13(3): 435-445.
5.	Tang Y, Tang W, Lin Y, et al. Combination of bone tissue engineering and BMP-2 gene transfection promotes bone healing in osteoporotic rats. Cell Biol Int, 2008, 32(9): 1150-1157.
6.	Kawabata K, Sakurai F, Koizumi N, et al. Adenovirus vector-mediated gene transfer into stem cells. Mol Pharm, 2006, 3(2): 95-103.
7.	胡炜, 俞兴, 徐林. 骨髓基质细胞诱导分化成骨方法及相关研究进展. 中国组织工程研究与临床康复, 2009, 13(1): 169-172.
8.	Niemeyer P, Krause U, Punzel M, et al. Mesenchymal stem cells for tissue engineering of bone: 3D-cultivation and osteogenic differentiation on mineralized collagen. Z Orthop Ihre Grenzgeb, 2003, 141(6): 712-717.
9.	Taira M, Nakao H, Takahashi J, et al. Effects of two vitamins, two growth factors and dexamethasone on the proliferation of rat bone marrow stromal cells and osteoblastic MC3T3-E1cells. J Oral Rehabil, 2003, 30(7): 697-701.
10.	Rodríguez JP, González M, Ríos S, et al. Cytoskeletal organization of human mesenchymal stem cells (MSC) changes during their osteogenic differentiation. J Cell Biochem, 2004, 93(4): 721-731.
11.	Otsuka E, Yamaguchi A, Shigehisa H, et al. Characterizations of osteoblastic differentiation of stromalcell line ST2 that is induced by ascorbic acid. Am J Physiol, 1999, 277(1 Pt 1): C132-138.
12.	Egusa H, Iida K, Kobayashi M, et al. Downregulation of extracellular matrix-related gene clusters during osteogenic differentiation of human bone marrow- and adipose tissue-derived stromal cells. Tissue Eng, 2007, 13(10): 2589-2600.
13.	Timmer NM, van Horssen J, Otte-Holler I, et al. Amyloid beta induces cellular relocalization and production of agrin and glypican-1. Brain Res, 2009, 1260: 38-46.
14.	Oreffo RO, Cooper C, Mason C, et al. Mesenchymal stem cells: lineage, plasticity, and skeletal therapeutic potential. Stem Cell Rev, 2005, 1(2): 169-178.
15.	Kimelman N, Pelled G, Gazit Z, et al. Applications of gene therapy and adult stem cells in bone bioengineering. Regen Med, 2006, 1(4): 549-561.
16.	Gugala Z, Olmsted-Davis EA, Gannon FH, et al. Osteo-induction by ex vivo adenovirus-mediated BMP-2 delivery is independent of cell type. Gene Ther, 2003, 10(16): 1289-1296.
17.	Gugala Z, Davis AR, Fouletier-Dilling CM, et al. Adenovirus BMP-2 induced osteogenesis in combination with collagen carriers. Biomaterials, 2007, 28(30): 4469-4479.
18.	Sipos W, Pietschmann P, Rauner M. Strategies for novel therapeutic approaches targeting cytokines and signaling pathways of osteoclasto- and osteoblastogenesis in the fight against immune-mediated bone and joint diseases. Curr Med Chem, 2008, 15(2): 127-136.
19.	Alcaraz MJ, Megias J, Garcia-Arnandis I, et al. New molecular targets for the treatment of osteoarthritis. Biochem Pharmacol, 2010, 80(1): 13-21.
20.	Fulci V, Scappucci G, Sebastiani GD, et al. miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis. Hum Immunol, 2010, 71(2): 206-211.
21.	Luo X, Tsai LM, Shen N, et al. Evidence for microRNA-mediated regulation in rheumatic diseases. Ann Rheum Dis, 2010, 69 Suppl 1: i30-36.
22.	Miyaki S, Sato T, Inoue A, et al. MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev, 2010, 24(11): 1173-1185.
23.	Babkina IV, Osipov DA, Solovyov YN, et al. Endostatin, placental growth factor, and fibroblast growth factors-1 and -2 in the sera of patients with primary osteosarcomas. Bull Exp Biol Med, 2009, 148(2): 246-249.