miR-93-5P SUPPRESSES OSTEOGENIC DIFFERENTIATION OF MOUSE C3H10T1/2 CELLS BY TARGETING Smad5_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

XULian ¹ , LIXiaolong ¹ , LIUYin ¹ ,  KONGQingquan ¹ , LONGDan ² , LIShengfu ²

1. Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;
2. Key Laboratory of Transplant Engineering and Immunology, Ministry of Healthy, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China;

Corresponding?author：

KONGQingquan, Email: kqqspine@126.com

Keywords：

C3H10T1/2 cells; miR-93-5p; Smad5; Mesenchymal stem cells; Osteogenic differentiation; Mouse

DOI：

10.7507/1002-1892.20150279

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Objective To investigate whether miR-93-5p suppresses osteogenic differentiation of mouse mesenchymal stem cells (C3H10T1/2) by targeting Smad5, a predicted target in silicon. Methods Smad5 3'-UTRluciferase vector (pmiR-RB-REPORT^TM) was constructed and dual-luciferase reporter gene assay was employed to examine the effect of miR-93-5p on Smad5 3'-UTR-luciferase activity to identify whether Smad5 was the target gene of miR-93-5p. miR-93-5p mimics (group M), miR-93-5p inhibitor (group In), miR-93-5p mimics negative control (group MC), and miR-93-5p inhibitor negative control (group InC) were transfected into the C3H10T1/2 cells, respectively, and followed by induction of osteogenic differentiation. After 48 hours, the real-time fluorescent quantitative PCR (qRTPCR) and Western blot assays were performed to detect the relative expressions of Smad5 mRNA and protein. At 14 days, to realize the regulation role of miR-93-5p in osteogenic differentiation, the extracellular calcium deposition during the osteogenesis of C3H10T1/2 cells was tested by Alizarin red staining. Results Dual-luciferase reporter gene assay showed that miR-93-5p could combine with Smad5 mRNA 3'-UTR specificity, and inhibited its luciferase activity (P<0.05). After 48 hours, no significant difference was shown in the relative expression of Smad5 mRNA between group M and group MC as well as between group In and group InC by qRT-PCR assay (P>0.05); however, the results of Western blot assay showed that the relative expression of Smad5 protein was significantly decreased in group M and increased in group In when compared with groups MC and InC (P<0.05). At 14 days after osteogenic induction, Alizarin red staining showed that the extracellular calcium deposition of group M was obviously less than that of group MC, and it was obviously more in group In than in group InC. Conclusion Smad5 may be the target gene of miR-93-5p. And miR-93-5p can suppress osteogenic differentiation of C3H10T1/2 cells by directly targeting Smad5.

Citation： XULian, LIXiaolong, LIUYin, KONGQingquan, LONGDan, LIShengfu. miR-93-5P SUPPRESSES OSTEOGENIC DIFFERENTIATION OF MOUSE C3H10T1/2 CELLS BY TARGETING Smad5. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(10): 1288-1294. doi: 10.7507/1002-1892.20150279 Copy

1.	Ding DC, Shyu WC, Lin SZ. Mesenchymal stem cells. Cell Transplant, 2011, 20(1):5-14.
2.	Wang S, Kawashima N, Sakamoto K, et al. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj. Biochem Biophys Res Commun, 2010, 400(1):39-45.
3.	Green DW, Kwon HJ, Jung HS. Osteogenic potency of nacre on human mesenchymal stem cells. Mol Cells, 2015, 38(3):267-272.
4.	Lau PW, MacRae IJ. The molecular machines that mediate microRNA maturation. J Cell Mol Med, 2009, 13(1):54-60.
5.	Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res, 2009, 19(7):1175-1183.
6.	Liu X, Fortin K, Mourelatos Z. MicroRNAs:biogenes is and molecular functions. Brain Pathol, 2008, 18(1):113-121.
7.	Mallanna SK, Rizzino A. Emergingroles of microRNAs in the control of embryonic stem cells and the genera-tion of induced pluripotent stem cells. Dev Biol, 2010, 344(1):16-25.
8.	Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development. Science, 2005, 309(5732):310-311.
9.	Jia W, Chen W, Kang J. The functions of microRNAs and long noncoding RNAs in embryonic and induced pluripotent stem cells. Genomics Proteomics Bioinformatics, 2013, 11(5):275-283.
10.	Bouyssou JM, Manier S, Huynh D, et al. Regulation of microRNAs in cancer metastasis. Biochim Biophys Acta, 2014, 1845(2):255-265.
11.	Liu C, Tang DG. MicroRNA regulation of cancer stem cells. Cancer Res, 2011, 71(18):5950-5954.
12.	Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci, 2008, 105(37):13906-13911.
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14.	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.
15.	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.
16.	康夏, 康菲, 楊波, 等. 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.
17.	Hwang S, Park SK, Lee HY, et al. miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells. FEBS Lett, 2014, 588(17):2957-2963.
18.	Huang K, Fu J, Zhou W, et al. MicroRNA-125b regulates osteogenic differentiation of mesenchymal stem cells by targeting Cbfβ in vitro. Biochimie, 2014, 102:47-55.
19.	徐練, 孔清泉. 調控成骨細胞分化及骨形成關鍵信號通路的研究進展. 中國修復重建外科雜志, 2014, 28(12):1484-1489.
20.	Hariharan R, Pillai MR. Homology modeling of the DNA-binding domain of human Smad5:A molecular model for inhibitor design. J Mol Graph Model, 2006, 24(4):271-277.
21.	Tao S, Sampath K. Alternative splicing of SMADs in differentiation and tissue homeostasis. Dev Growth Differ, 2010, 52(4):335-342.
22.	Nishimura R, Kato Y, Chen D, et al. Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12. J Biol Chem, 1998, 273(4):1872-1879.
23.	Zhu S, Wu H, Wu F, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res, 2008, 18(3):350-359.
24.	Srivastava S, Kumar N, Roy P. Role of ERK/NFκB in vanadium (IV) oxide mediated osteoblast differentiation in C3H10t1/2 cells. Biochimie, 2014, 101:132-144.

1. Ding DC, Shyu WC, Lin SZ. Mesenchymal stem cells. Cell Transplant, 2011, 20(1):5-14.
2. Wang S, Kawashima N, Sakamoto K, et al. Osteogenic differentiation of mouse mesenchymal progenitor cell, Kusa-A1 is promoted by mammalian transcriptional repressor Rbpj. Biochem Biophys Res Commun, 2010, 400(1):39-45.
3. Green DW, Kwon HJ, Jung HS. Osteogenic potency of nacre on human mesenchymal stem cells. Mol Cells, 2015, 38(3):267-272.
4. Lau PW, MacRae IJ. The molecular machines that mediate microRNA maturation. J Cell Mol Med, 2009, 13(1):54-60.
5. Lee I, Ajay SS, Yook JI, et al. New class of microRNA targets containing simultaneous 5'-UTR and 3'-UTR interaction sites. Genome Res, 2009, 19(7):1175-1183.
6. Liu X, Fortin K, Mourelatos Z. MicroRNAs:biogenes is and molecular functions. Brain Pathol, 2008, 18(1):113-121.
7. Mallanna SK, Rizzino A. Emergingroles of microRNAs in the control of embryonic stem cells and the genera-tion of induced pluripotent stem cells. Dev Biol, 2010, 344(1):16-25.
8. Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development. Science, 2005, 309(5732):310-311.
9. Jia W, Chen W, Kang J. The functions of microRNAs and long noncoding RNAs in embryonic and induced pluripotent stem cells. Genomics Proteomics Bioinformatics, 2013, 11(5):275-283.
10. Bouyssou JM, Manier S, Huynh D, et al. Regulation of microRNAs in cancer metastasis. Biochim Biophys Acta, 2014, 1845(2):255-265.
11. Liu C, Tang DG. MicroRNA regulation of cancer stem cells. Cancer Res, 2011, 71(18):5950-5954.
12. Li Z, Hassan MQ, Volinia S, et al. A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci, 2008, 105(37):13906-13911.
13. 幸嶸, 孔清泉, 項舟, 等. 小鼠干細胞成骨和成軟骨分化特異性微小RNA的初步研究. 中國修復重建外科雜志, 2014, 28(8):1009-1016.
14. 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.
15. 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.
16. 康夏, 康菲, 楊波, 等. 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.
17. Hwang S, Park SK, Lee HY, et al. miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells. FEBS Lett, 2014, 588(17):2957-2963.
18. Huang K, Fu J, Zhou W, et al. MicroRNA-125b regulates osteogenic differentiation of mesenchymal stem cells by targeting Cbfβ in vitro. Biochimie, 2014, 102:47-55.
19. 徐練, 孔清泉. 調控成骨細胞分化及骨形成關鍵信號通路的研究進展. 中國修復重建外科雜志, 2014, 28(12):1484-1489.
20. Hariharan R, Pillai MR. Homology modeling of the DNA-binding domain of human Smad5:A molecular model for inhibitor design. J Mol Graph Model, 2006, 24(4):271-277.
21. Tao S, Sampath K. Alternative splicing of SMADs in differentiation and tissue homeostasis. Dev Growth Differ, 2010, 52(4):335-342.
22. Nishimura R, Kato Y, Chen D, et al. Smad5 and DPC4 are key molecules in mediating BMP-2-induced osteoblastic differentiation of the pluripotent mesenchymal precursor cell line C2C12. J Biol Chem, 1998, 273(4):1872-1879.
23. Zhu S, Wu H, Wu F, et al. MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res, 2008, 18(3):350-359.
24. Srivastava S, Kumar N, Roy P. Role of ERK/NFκB in vanadium (IV) oxide mediated osteoblast differentiation in C3H10t1/2 cells. Biochimie, 2014, 101:132-144.

Chinese Journal of Reparative and Reconstructive Surgery

miR-93-5P SUPPRESSES OSTEOGENIC DIFFERENTIATION OF MOUSE C3H10T1/2 CELLS BY TARGETING Smad5

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