CHONDROGENESIS-SPECIFIC MICRORNA EXPRESSION PATTERN ANALYSIS IN CHONDROGENIC DIFFERENTIATION OF HUMAN ADIPOSE-DERIVED STEM CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

ZHANGZiji ,  KANGYan , ZHANGZhiqi , YANGZibo , FANGShuying , SHENGPuyi , HEAishan , FUMing , LIAOWeiming

Department of Joint Surgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou Guangdong, 510080, P. R. China;

Corresponding author：

KANGYan, Email: neokang@163.com

Keywords：

MicroRNA; Human adipose-derived stem cells; Chondrogenic differentiation; Microarray assay; Target gene

DOI：

10.7507/1002-1892.20150017

Video：

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Objective To investigate the microRNA (miRNA) expression profile during chondrogenic differentiation of human adipose-derived stem cells (hADSCs), and assess the roles of involved miRNAs during chondrogenesis. Methods hADSCs were harvested and cultured from donors who underwent elective liposuction or other abdominal surgery. When the cells were passaged to P3, chondrogenic induction medium was used for chondrogenic differentiation. The morphology of the cells was observed by inverted phase contrast microscopy. Alcian blue staining was carried out at 21 days after induction to access the chondrogenic status. The expressions of chondrogenic proteins were detected by ELISA at 0, 7, 14, and 21 days. The miRNA expression profiles at pre- and post-chondrogenic induction were obtained by microarray assay, and differentially expressed miRNAs were verified by real-time quantitative PCR (qRT-PCR). The targets of the miRNAs were predicted by online software programs. Results hADSCs were cultured successfully and induced with chondrogenic medium. At 21 days after chondrogenic induction, the cells were stained positively for alcian blue staining. At 7, 14, and 21 days after chondrogenic induction, the levels of collogen type Ⅱ, Col2a1, aggrecan, Col10a1, and chondroitin sulfate in induced hADSCs were significantly higher than those in noninduced hADSCs (P<0.05). Eleven differentially expressed miRNAs were found, including seven up-regulated and four down-regulated. Predicted target genes of the differentially expressed miRNAs were based on the overlap from three public prediction algorithms, with the known functions of regulating chondrogenic differentiation of stem cells, selfrenewal, signal transduction, intracellular signaling cascade, and cell cycle control. Conclusion A group of miRNAs and their target genes are identified, which may play important roles in regulating chondrogenic differentiation of hADSCs. These results will facilitate the initial understanding of the molecular mechanism of chondrogenic differentiation in hADSCs and subsequently control hADSCs differentiation, and provide high performance seed cells for cartilage tissue engineering.

Citation： ZHANGZiji, KANGYan, ZHANGZhiqi, YANGZibo, FANGShuying, SHENGPuyi, HEAishan, FUMing, LIAOWeiming. CHONDROGENESIS-SPECIFIC MICRORNA EXPRESSION PATTERN ANALYSIS IN CHONDROGENIC DIFFERENTIATION OF HUMAN ADIPOSE-DERIVED STEM CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2015, 29(1): 74-80. doi: 10.7507/1002-1892.20150017 Copy

1.	Nakao N, Nakayama T, Yahata T, et al. Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in vitro and in vivo:advantages over bone marrow-derived mesenchymal stem cells. Am J Pathol, 2010, 177(2):547-554.
2.	Kim D, Monaco E, Maki A, et al. Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels. Cell Tissue Res, 2010, 341(3):359-370.
3.	Brennecke J, Hipfner DR, Stark A, et al. Bantamen codes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell, 2003, 113(1):25-36.
4.	Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineage differentiation. Science, 2004, 303(5654):83-86.
5.	Dostie J, Mourelatos Z, Yang M, et al. Numerous microRNPs in neuronal cells containing novel microRNAs. RNA, 2003, 9(2):180-186.
6.	Lü J, Qian J, Chen F, et al. Differential expression of components of the microRNA machinery during mouse organogenesis. Biochem Biophys Res Commun, 2005, 334(2):319-323.
7.	Goldring MB, Otero M. Inflammation in osteoarthritis. Curr Opin Rheumatol, 2011, 23(5):471-478.
8.	Dong S, Yang B, Guo H, et al. MicroRNAs regulate osteogenesis and chondrogenesis. Biochem Biophys Res Commun, 2012, 418(4):587-591.
9.	Dominici MI, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4):315-317.
10.	Zhang ZJ, Zhang H, Kang Y, et al. miRNA expression profile during osteogenic differentiation of human adipose-derived stem cells. J Cell Biochem, 2012, 113(3):888-898.
11.	Miyaki S, Nakasa T, Otsuki S, et al. MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses. Arthritis Rheum, 2009, 60(9):2723-2730.
12.	Yamasaki K, Nakasa T, Miyaki S, et al. Expression of MicroRNA-146a in osteoarthritis cartilage. Arthritis Rheum, 2009, 60(4):1035-1041.
13.	Kobayashi T, Lu J, Cobb BS, et al. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc Natl Acad Sci U S A, 2008, 105(6):1949-1954.
14.	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.
15.	Berezikov E, Guryev V, van de Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell, 2005, 120(1):21-24.
16.	Han J, Yang T, Gao J, et al. Specific microRNA expression during chondrogenesis of human mesenchymal stem cells. Int J Mol Med, 2010, 25(3):377-384.
17.	Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development. Science, 2005, 309(5732):310-311.
18.	Tuddenham L, Wheeler G, Ntounia-Fousara S, et al. The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett, 2006, 580(17):4214-4217.
19.	Iliopoulos D, Malizos KN, Oikonomou P, et al. Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks. PLoS One, 2008, 3(11):e3740.
20.	Xu J, Kang Y, Liao WM, et al. MiR-194 regulates chondrogenic differentiation of human adipose-derived stem cells by targeting Sox5. PLoS One, 2012, 7(3):e31861.
21.	Hattori T, Coustry F, Stephens S, et al. Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5. Nucleic Acids Res, 2008, 36(9):3011-3024.
22.	Zhang Z, Xing X, Hensley G, et al. Resistin induces expression of proinflammatory cytokines and chemokines in human articular chondrocytes via transcription and mRNA stabilization. Arthritis Rheum, 2010, 62(7):1993-2003.
23.	Zhang Z, Bryan JL, DeLassus E, et al. CCAAT/enhancer-binding protein β and NF-κB mediate high level expression of chemokine genes CCL3 and CCL4 by human chondrocytes in response to IL-1β. J Biol Chem, 2010, 285(43):33092-33103.
24.	Wehling N, Palmer GD, Pilapil C, et al. Interleukin-1 beta and tumor necrosis factor alpha inhibit chondrogenesis by human mesenchymal stromal cells through NF-kappaB-dependent pathways. Arthritis Rheum, 2009, 60(3):801-812.
25.	Han Y, Chen J, Zhao X, et al. MicroRNA expression signatures of bladder cancer revealed by deep sequencing. PLoS One, 2011, 6(3):e18286.

1. Nakao N, Nakayama T, Yahata T, et al. Adipose tissue-derived mesenchymal stem cells facilitate hematopoiesis in vitro and in vivo:advantages over bone marrow-derived mesenchymal stem cells. Am J Pathol, 2010, 177(2):547-554.
2. Kim D, Monaco E, Maki A, et al. Morphologic and transcriptomic comparison of adipose- and bone-marrow-derived porcine stem cells cultured in alginate hydrogels. Cell Tissue Res, 2010, 341(3):359-370.
3. Brennecke J, Hipfner DR, Stark A, et al. Bantamen codes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell, 2003, 113(1):25-36.
4. Chen CZ, Li L, Lodish HF, et al. MicroRNAs modulate hematopoietic lineage differentiation. Science, 2004, 303(5654):83-86.
5. Dostie J, Mourelatos Z, Yang M, et al. Numerous microRNPs in neuronal cells containing novel microRNAs. RNA, 2003, 9(2):180-186.
6. Lü J, Qian J, Chen F, et al. Differential expression of components of the microRNA machinery during mouse organogenesis. Biochem Biophys Res Commun, 2005, 334(2):319-323.
7. Goldring MB, Otero M. Inflammation in osteoarthritis. Curr Opin Rheumatol, 2011, 23(5):471-478.
8. Dong S, Yang B, Guo H, et al. MicroRNAs regulate osteogenesis and chondrogenesis. Biochem Biophys Res Commun, 2012, 418(4):587-591.
9. Dominici MI, Le Blanc K, Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006, 8(4):315-317.
10. Zhang ZJ, Zhang H, Kang Y, et al. miRNA expression profile during osteogenic differentiation of human adipose-derived stem cells. J Cell Biochem, 2012, 113(3):888-898.
11. Miyaki S, Nakasa T, Otsuki S, et al. MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses. Arthritis Rheum, 2009, 60(9):2723-2730.
12. Yamasaki K, Nakasa T, Miyaki S, et al. Expression of MicroRNA-146a in osteoarthritis cartilage. Arthritis Rheum, 2009, 60(4):1035-1041.
13. Kobayashi T, Lu J, Cobb BS, et al. Dicer-dependent pathways regulate chondrocyte proliferation and differentiation. Proc Natl Acad Sci U S A, 2008, 105(6):1949-1954.
14. 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.
15. Berezikov E, Guryev V, van de Belt J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell, 2005, 120(1):21-24.
16. Han J, Yang T, Gao J, et al. Specific microRNA expression during chondrogenesis of human mesenchymal stem cells. Int J Mol Med, 2010, 25(3):377-384.
17. Wienholds E, Kloosterman WP, Miska E, et al. MicroRNA expression in zebrafish embryonic development. Science, 2005, 309(5732):310-311.
18. Tuddenham L, Wheeler G, Ntounia-Fousara S, et al. The cartilage specific microRNA-140 targets histone deacetylase 4 in mouse cells. FEBS Lett, 2006, 580(17):4214-4217.
19. Iliopoulos D, Malizos KN, Oikonomou P, et al. Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks. PLoS One, 2008, 3(11):e3740.
20. Xu J, Kang Y, Liao WM, et al. MiR-194 regulates chondrogenic differentiation of human adipose-derived stem cells by targeting Sox5. PLoS One, 2012, 7(3):e31861.
21. Hattori T, Coustry F, Stephens S, et al. Transcriptional regulation of chondrogenesis by coactivator Tip60 via chromatin association with Sox9 and Sox5. Nucleic Acids Res, 2008, 36(9):3011-3024.
22. Zhang Z, Xing X, Hensley G, et al. Resistin induces expression of proinflammatory cytokines and chemokines in human articular chondrocytes via transcription and mRNA stabilization. Arthritis Rheum, 2010, 62(7):1993-2003.
23. Zhang Z, Bryan JL, DeLassus E, et al. CCAAT/enhancer-binding protein β and NF-κB mediate high level expression of chemokine genes CCL3 and CCL4 by human chondrocytes in response to IL-1β. J Biol Chem, 2010, 285(43):33092-33103.
24. Wehling N, Palmer GD, Pilapil C, et al. Interleukin-1 beta and tumor necrosis factor alpha inhibit chondrogenesis by human mesenchymal stromal cells through NF-kappaB-dependent pathways. Arthritis Rheum, 2009, 60(3):801-812.
25. Han Y, Chen J, Zhao X, et al. MicroRNA expression signatures of bladder cancer revealed by deep sequencing. PLoS One, 2011, 6(3):e18286.

Chinese Journal of Reparative and Reconstructive Surgery

CHONDROGENESIS-SPECIFIC MICRORNA EXPRESSION PATTERN ANALYSIS IN CHONDROGENIC DIFFERENTIATION OF HUMAN ADIPOSE-DERIVED STEM CELLS

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