PRELIMINARY STUDY ON microRNA REGULATED OSTEOGENIC AND CHONDROGENIC DIFFERENTIATION OF MOUSE STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XINGRong ¹ ,  KONGQingquan ¹ , XIANGZhou ¹ , YANGJing ¹ , LUOJingcong ² , DENGLi ² , XIEHuiqi ²

1. Department of Orthopaedics, Sichuan University, Chengdu Sichuan, 610041, P. R. China;
2. Laboratory of Stem Cells and Tissue Engineering, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;

Corresponding author：

KONGQingquan, Email: kongspine@126.com

Keywords：

C3H10T1/2 cells; Osteogenic differentiation; Chondrogenic differentiation; microRNA

DOI：

10.7507/1002-1892.20140222

Video：

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Objective To investigate the specific microRNA (miRNA) in osteogenic and chondrogenic differentiations of C3H10T1/2 cells. Methods C3H10T1/2 cells were induced to differentiate into osteoblasts and chondrocytes.Specific miRNA more than 2 fold change and 2 average normalized probe signal between C3H10T1/2 and C3H10T1/2-derived osteoblast,and between C3H10T1/2 and C3H10T1/2-derived chondrocytes were screened out by miRNA microarray,and verified by real-time fluorescence quantitative PCR (RT-qPCR). Results Alkaline phosphatase expression of osteogenic induced group was significantly higher than that of control group at 7 days after induced (P<0.05).RT-qPCR results showed the expressions of Runx2,serine protease (Sp7),collagen type I,and osteopontin (OPN) genes were significantly increased at 7,14,and 21 days after induced when compared with before induced (P<0.05).Western blot results showed the expressions of Runx2,Sp7,collagen type I,and OPN proteins of osteogenic induced group were significantly higher than those of control group at 21 days after induced (P<0.05).The expressions of SOX9,collagen type Ⅱ,Aggrecan,and Has2 were significantly increased at 5,10,and 15 days after induced when compared with before induced (P<0.05).The expressions of SOX9,collagen type 2,Aggrecan,and Has2 proteins of chondrogenic induced group were significantly higher than those of control group at 15 days after induced (P<0.05).Totally,10 osteogenic and 3 chondrogenic miRNA more than 2 fold change and 2 average normalized probe signal were screened out by miRNA microarray.RT-qPCR results of these specific miRNAs were similar to microarray results except miR-455-3p. Conclusion Specific miRNAs are screened out by microarray and it is a good foundation for the future study on miRNA functional verification and target gene prediction.

Citation： XINGRong, KONGQingquan, XIANGZhou, YANGJing, LUOJingcong, DENGLi, XIEHuiqi. PRELIMINARY STUDY ON microRNA REGULATED OSTEOGENIC AND CHONDROGENIC DIFFERENTIATION OF MOUSE STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(8): 1009-1016. doi: 10.7507/1002-1892.20140222 Copy

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2.	Vaucheret H.Post-transcriptional small RNA pathways in plants:mechanisms and regulations.Genes Dev,2006,20(7):759-771.
3.	Du T,Zamore PD.microPrimer:the biogenesis and function of microRNA.Development,2005,132(21):4645-4652.
4.	Ying SY,Lin SL.Intron-derived microRNAs-fine tuning of gene functions.Gene,2004,342(1):25-28.
5.	Breving K,Esquela-Kerscher A.The complexities of microRNA regulation mirandering around the rules.Int J Biochem Cell Biol,2010,42(8):1316-1329.
6.	Hata A,Davis BN.Control of microRNA biogenesis by TGFbeta signaling pathway-A novel role of Smads in the nucleus.Cytokine Growth Factor Rev,2009,20(5-6):517-521.
7.	Gangaraju VK,Lin H.MicroRNAs:key regulators of stem cells.Nat Rev Mol Cell Biol,2009,10(2):116-125.
8.	Li Z,Hassan MQ,Jafferji M,et al.Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation.J Biol Chem,2009,284(23):15676-15684.
9.	Hoffmann A,Pelled G,Turgeman G,et al.Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells.J Clin Invest,2006,116(4):940-952.
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11.	Wang M,Su Y,Sun H,et al.Induced endothelial differentiation of cells from a murine embryonic mesenchymal cell line C3H/10T1/2 by angiogenic factors in vitro.Differentiation,2010,79(1):21-30.
12.	Kapinas K,Kessler CB,Delany AM.miR-29 suppression of osteonectin in osteoblasts:regulation during differentiation and by canonical Wnt signaling.J Cell Biochem,2009,108(1):216-224.
13.	Kapinas K,Kessler C,Ricks T,et al.miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop.J Biol Chem,2010,285(33):25221-25231.
14.	Zhang J,Tu Q,Bonewald LF,et al.Effects of miR-335-5p in modulating osteogenic differentiation by specifically downregulating Wnt antagonist DKK1.J Bone Miner Res,2011,26(8):1953-1963.
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19.	Wienholds E,Kloosterman WP,Miska E,et al.MicroRNA expression in zebrafish embryonic development.Science,2005,309(5732):310-311.
20.	Song B,Wang Y,Xi Y,et al.Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and coloncancer cells.Oncogene,2009,28(46):4065-4074.
21.	Miyaki S,Sato T,Inoue A,et al.MicroRNA-140 plays dualroles in both cartilage development and homeostasis.Genes Dev,2010,24(11):1173-1185.
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24.	Song J,Kim D,Jin EJ.MicroRNA-488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells.Cell Biol Int,2011,35(2):179-185.
25.	Yang B,Guo H,Zhang Y,et al.The microRNA expression profiles of mouse mesenchymal stem cell during chondrogenic differentiation.BMB Rep,2011,44(1):28-33.
26.	Martinez-Sanchez A,Dudek KA,Murphy CL.Regulation of human chondrocyte function through directin hibition of cartilage master-regulator SOX9 by miRNA-145.J Biol Chem,2012,287(2):916-924.
27.	Dudek KA,Lafont JE,Martinez-Sanchez A,et al.Type Ⅱ collagen expression is regulated by tissue-specific miR-675 in human articular chondrocytes.J Biol Chem,2010,285(32):24381-24387.
28.	Kongcharoensombat W,Nakasa T,Ishikawa M,et al.The effect of microRNA-21 on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel.Knee Surg SportsTraumatol Arthrosc,2010,18(12):1679-1684.
29.	Yamasaki K,Nakasa T,Miyaki S,et al.Expression of microRNA-146a in osteoarthritis cartilage.Arthritis Rheum,2009,60(4):1035-1041.
30.	Paik S,Jung HS,Lee S,et al.miR-449a regulates the chondrogenesis of human mesenchymal stem cells through direct targeting of lymphoid enhancer-binding factor-1.Stem Cells Dev,2012,21(18):3298-3308.
31.	Wang T,Xu Z.miR-27 promotes osteoblast differentiation by modulating Wnt signaling.Biochem Biophys Res Commun,2010,402:186-189.
32.	Luzi E,Marini F,Sala SC,et al.Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor.J Bone Miner Res,2008,23(2):287-295.
33.	Guo CJ,Pan Q,Li DG,et al.miR-15b and miR-16 are implicated in activation of the rat hepatic stellate cell:An essential role for apoptosis.J Hepatol,2009,50(4):766-778.
34.	Zhang ZW,Zhang LQ,Ding L,et al.MicroRNA-19b downregulates insulin 1 through targeting transcription factor NeuroD1.FEBS Letters,2011,585(16):2592-2598.
35.	Swingler TE,Wheeler G,Carmont V,et al.The expression and function of microRNAs in chondrogenesis and osteoarthritis.Arthritis Rheum,2012,64(6):1909-1919.

1. Hwang HW,Mendell JT.MicroRNAs in cell proliferation cell death and tumorigenesis.Br J Cancer,2006,94(6):776-780.
2. Vaucheret H.Post-transcriptional small RNA pathways in plants:mechanisms and regulations.Genes Dev,2006,20(7):759-771.
3. Du T,Zamore PD.microPrimer:the biogenesis and function of microRNA.Development,2005,132(21):4645-4652.
4. Ying SY,Lin SL.Intron-derived microRNAs-fine tuning of gene functions.Gene,2004,342(1):25-28.
5. Breving K,Esquela-Kerscher A.The complexities of microRNA regulation mirandering around the rules.Int J Biochem Cell Biol,2010,42(8):1316-1329.
6. Hata A,Davis BN.Control of microRNA biogenesis by TGFbeta signaling pathway-A novel role of Smads in the nucleus.Cytokine Growth Factor Rev,2009,20(5-6):517-521.
7. Gangaraju VK,Lin H.MicroRNAs:key regulators of stem cells.Nat Rev Mol Cell Biol,2009,10(2):116-125.
8. Li Z,Hassan MQ,Jafferji M,et al.Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation.J Biol Chem,2009,284(23):15676-15684.
9. Hoffmann A,Pelled G,Turgeman G,et al.Neotendon formation induced by manipulation of the Smad8 signalling pathway in mesenchymal stem cells.J Clin Invest,2006,116(4):940-952.
10. Taylor SM,Jones PA.Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine.Cell,1979,17(4):771-779.
11. Wang M,Su Y,Sun H,et al.Induced endothelial differentiation of cells from a murine embryonic mesenchymal cell line C3H/10T1/2 by angiogenic factors in vitro.Differentiation,2010,79(1):21-30.
12. Kapinas K,Kessler CB,Delany AM.miR-29 suppression of osteonectin in osteoblasts:regulation during differentiation and by canonical Wnt signaling.J Cell Biochem,2009,108(1):216-224.
13. Kapinas K,Kessler C,Ricks T,et al.miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop.J Biol Chem,2010,285(33):25221-25231.
14. Zhang J,Tu Q,Bonewald LF,et al.Effects of miR-335-5p in modulating osteogenic differentiation by specifically downregulating Wnt antagonist DKK1.J Bone Miner Res,2011,26(8):1953-1963.
15. Jeon EJ,Lee KY,Choi NS,et al.Bone morphogenetic protein-2 stimulates Runx2 acetylation.J Biol Chem,2006,281(24):16502-16511.
16. Hassan MQ,Gordon JA,Beloti MM,et al.A network connecting Runx2,SATB2,and the miR-23a~27a~24-2 cluster regulates the osteoblast differentiation program.Proc Natl Acad Sci U S A,2010,107(46):19879-19884.
17. He J,Zhang JF,Yi C,et al.miRNA-mediated functional changes through co-regulating function related genes.PLoS One,2010,5(10):e13558.
18. Mizuno Y,Yagi K,Tokuzawa Y,et al.miR-125b inhibits osteoblastic differentiation by down-regulation of cell proliferation.Biochem Biophys Res Commun,2008,368(2):267-272.
19. Wienholds E,Kloosterman WP,Miska E,et al.MicroRNA expression in zebrafish embryonic development.Science,2005,309(5732):310-311.
20. Song B,Wang Y,Xi Y,et al.Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and coloncancer cells.Oncogene,2009,28(46):4065-4074.
21. Miyaki S,Sato T,Inoue A,et al.MicroRNA-140 plays dualroles in both cartilage development and homeostasis.Genes Dev,2010,24(11):1173-1185.
22. Lin EA,Kong L,Bai XH,et al.miR-199a,a bone morphogenic protein 2-responsive MicroRNA,regulates chondrogenesis via direct targeting to Smad1.J Biol Chem,2009,284(17):11326-11335.
23. Ohgawara T,Kubota S,Kawaki H,et al.Regulation of chondrocytic phenotype by microRNA 18a:involvement of Ccn2/Ctgf as a major target gene.FEBS Lett,2009,583(6):1006-1010.
24. Song J,Kim D,Jin EJ.MicroRNA-488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells.Cell Biol Int,2011,35(2):179-185.
25. Yang B,Guo H,Zhang Y,et al.The microRNA expression profiles of mouse mesenchymal stem cell during chondrogenic differentiation.BMB Rep,2011,44(1):28-33.
26. Martinez-Sanchez A,Dudek KA,Murphy CL.Regulation of human chondrocyte function through directin hibition of cartilage master-regulator SOX9 by miRNA-145.J Biol Chem,2012,287(2):916-924.
27. Dudek KA,Lafont JE,Martinez-Sanchez A,et al.Type Ⅱ collagen expression is regulated by tissue-specific miR-675 in human articular chondrocytes.J Biol Chem,2010,285(32):24381-24387.
28. Kongcharoensombat W,Nakasa T,Ishikawa M,et al.The effect of microRNA-21 on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel.Knee Surg SportsTraumatol Arthrosc,2010,18(12):1679-1684.
29. Yamasaki K,Nakasa T,Miyaki S,et al.Expression of microRNA-146a in osteoarthritis cartilage.Arthritis Rheum,2009,60(4):1035-1041.
30. Paik S,Jung HS,Lee S,et al.miR-449a regulates the chondrogenesis of human mesenchymal stem cells through direct targeting of lymphoid enhancer-binding factor-1.Stem Cells Dev,2012,21(18):3298-3308.
31. Wang T,Xu Z.miR-27 promotes osteoblast differentiation by modulating Wnt signaling.Biochem Biophys Res Commun,2010,402:186-189.
32. Luzi E,Marini F,Sala SC,et al.Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor.J Bone Miner Res,2008,23(2):287-295.
33. Guo CJ,Pan Q,Li DG,et al.miR-15b and miR-16 are implicated in activation of the rat hepatic stellate cell:An essential role for apoptosis.J Hepatol,2009,50(4):766-778.
34. Zhang ZW,Zhang LQ,Ding L,et al.MicroRNA-19b downregulates insulin 1 through targeting transcription factor NeuroD1.FEBS Letters,2011,585(16):2592-2598.
35. Swingler TE,Wheeler G,Carmont V,et al.The expression and function of microRNAs in chondrogenesis and osteoarthritis.Arthritis Rheum,2012,64(6):1909-1919.

Chinese Journal of Reparative and Reconstructive Surgery

PRELIMINARY STUDY ON microRNA REGULATED OSTEOGENIC AND CHONDROGENIC DIFFERENTIATION OF MOUSE STEM CELLS

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