Role of Spry1 in osteogenic differentiation of human bone marrow mesenchymal stem cells under miR-21 regulation

doi:10.3969/j.issn.2095-4344.2014.32.001

Abstract

Abstract:

BACKGROUND: Previous studies have found that miR-21 expression is increased during osteogenic differentiation of bone marrow mesenchymal stem cells, but the action and molecular mechanism of miR-21 are still unclear.
OBJECTIVE: To verify the target gene of miR-21, Spry1, and to explore the role of Spry1 in osteogenic differentiation of human bone marrow mesenchymal stem cells.
METHODS: Luciferase report was used to verify Spry1 gene targeted by miR-21, and western blot assay was used to detect the expression of Spry1 in the osteogenesis of human bone marrow mesenchymal stem cells. Spry1 expression vector was established and transfected into human bone marrow mesenchymal stem cells. Osteogenesis ability of human bone marrow mesenchymal stem cells was analyzed after Spry1 high expression by alkaline phosphatase, alizarin red staining, RT-PCR and western blot.
RESULTS AND CONCLUSION: Luciferase report suggested that Spry1 was a target gene of miR-21. The expression level of Spry1 was decreased in the osteogenesis of human bone marrow mesenchymal stem cells. Increasing expression of Spry1 could inhibit osteogenic differentiation of human bone marrow mesenchymal stem cells. These results indicate that Spry1 as a target gene of miR-21 negatively regulates osteogenic differentiation
of human bone marrow mesenchymal stem cells, and plays an important role in bone formation process.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: bone marrow, mesenchymal stem cells, cell transdifferentiation, osteoblasts, microRNAs, gene expression

CLC Number:

R394.2

Yang Nan, Zhou Wei, Wang Guang, Ding Yin, Jin Yan. Role of Spry1 in osteogenic differentiation of human bone marrow mesenchymal stem cells under miR-21 regulation[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(32): 5085-5089.

Figures/Tables 1

2.1 荧光素酶报告系统验证Spry1为miR-21的靶基因为了验证miR-21是否通过与Spry1基因3’-UTR区结合抑制靶基因的表达，通过将扩增的Spry1基因3’-UTR片段插入pMiR-report多克隆位点构建了荧光素酶报告质粒。共转染荧光素酶报告质粒和pre-miR-21后，利用荧光素酶报告检测仪检测荧光素酶活性，发现荧光素酶活性明显下降。相反，共转染荧光素酶报告质粒和anti-miR-21后荧光素酶活性显著升高(图1A)。 2.2 miR-21能够降低Spry1蛋白表达为了进一步验证miR-21能在转录后水平抑制Spry1的表达，检测了不同miR-21表达水平下Spry1蛋白表达的变化。Western Blot结果提示，在人骨髓间充质干细胞中过表达miR-21能够显著降低Spry1的蛋白表达。相反，抑制miR-21表达后，能够促进Spry1蛋白的表达(图1B)。 2.3 Spry1在骨髓间充质干细胞成骨过程中的表达为了验证Spry1在人骨髓间充质干细胞成骨分化中的作用，首先检测了Spry1在人骨髓间充质干细胞成骨分化中的表达。结果发现，在成骨诱导第7天和第10天Spry1的表达显著下降，相反，miR-21在成骨7 d和10 d表达显著升高。这一结果说明在人骨髓间充质干细胞成骨分化过程中，miR-21可能负向调控Spry1表达(图2)。 2.4 Spry1在人骨髓间充质干细胞成骨分化中的作用为了进一步验证Spry1在人骨髓间充质干细胞成骨分化过程中的调控作用，实验构建了人Spry1的慢病毒表达载体。通过感染原代人骨髓间充质干细胞来高表达Spry1。同时构建慢病毒GFP表达载体作为对照，检测转染效率。并且，病毒感染2 d后提取RNA，real time RT-PCR 检测感染病毒后Spry1的表达。结果发现，感染Spry1慢病毒载体后，Spry1表达上升了5倍。确定Spry1高表达成功后，将稳定表达Spry1的细胞成骨诱导7 d和21 d，碱性磷酸酶和茜素红染色观察人骨髓间充质干细胞成骨分化能力。结果发现，Spry1高表达组人骨髓间充质干细胞活性降低，茜素红染色显示钙结节明显减少(图3A)。为了进一步确定Spry1对成骨分化的抑制作用，人骨髓间充质干细胞成骨诱导7 d后提取RNA和蛋白，检测成骨特异性基因Runx2和Osterix的表达。结果显示Spry1高表达后能够显著降低Runx2，Osterix的基因和蛋白的表达(图3B，C)。以上结果证明，Spry1负向调控人骨髓间充质干细胞体外成骨分化。"

References

[1] Raucci MG, Guarino V, Ambrosio L.Biomimetic strategies for bone repair and regeneration.J Funct Biomater. 2012;3(3): 688-705.
[2] Hakimi M, Grassmann JP, Betsch M,et al.The Composite of Bone Marrow Concentrate and PRP as an Alternative to Autologous Bone Grafting.PLoS One. 2014;9(6):e100143.
[3] Huri PY, Arda Ozilgen B, Hutton DL,et al. Scaffold pore size modulates in vitro osteogenesis of human adipose-derived stem/stromal cells.Biomed Mater. 2014;9(4):045003.
[4] Xu HF, Ding YJ, Zhang ZX,et al. MicroRNA?21 regulation of the progression of viral myocarditis to dilated cardiomyopathy. Mol Med Rep. 2014;10(1):161-168.
[5] Wang L, He L, Zhang R, et al. Regulation of T lymphocyte activation by microRNA-21.Mol Immunol. 2014;59(2): 163-171.
[6] Mekkawy AH, Pourgholami MH, Morris DL.Human Sprouty1 suppresses growth, migration, and invasion in human breast cancer cells.Tumour Biol. 2014;35(5):5037-5048.
[7] Cheung KS, Sposito N, Stumpf PS,et al. MicroRNA-146a Regulates Human Foetal Femur Derived Skeletal Stem Cell Differentiation by Down-Regulating SMAD2 and SMAD3. PLoS One. 2014 ;9(6):e98063.
[8] Gao J, Yang T, Han J, et al. MicroRNA expression during osteogenic differentiation of human multipotent mesenchymal stromal cells from bone marrow.J Cell Biochem. 2011;112(7): 1844-1856.
[9] Hanafusa H, Torii S, Yasunaga T,et al. Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway.Nat Cell Biol. 2002;4(11):850-858.
[10] Li H, Xie H, Liu W,et al. A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans.J Clin Invest. 2009;119(12): 3666-3677.
[11] Ng F, Boucher S, Koh S,et al. PDGF, TGF-beta, and FGF signaling is important for differentiation and growth of mesenchymal stem cells (MSCs): transcriptional profiling can identify markers and signaling pathways important in differentiation of MSCs into adipogenic, chondrogenic, and osteogenic lineages.Blood. 2008;112(2):295-307.
[12] Thum T, Gross C, Fiedler J,et al. MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signalling in fibroblasts.Nature. 2008;456(7224):980-984.
[13] Reich A, Sapir A, Shilo B.Sprouty is a general inhibitor of receptor tyrosine kinase signaling.Development. 1999;126 (18):4139-4147.
[14] Gross I, Bassit B, Benezra M,et al. Mammalian sprouty proteins inhibit cell growth and differentiation by preventing ras activation.J Biol Chem. 2001;276(49):46460-46468.
[15] Shea KL, Xiang W, LaPorta VS,et al. Sprouty1 regulates reversible quiescence of a self-renewing adult muscle stem cell pool during regeneration.Cell Stem Cell. 2010;6(2): 117-129.
[16] Basson MA, Akbulut S, Watson-Johnson J,et al. Sprouty1 is a critical regulator of GDNF/RET-mediated kidney induction. Dev Cell. 2005;8(2):229-239.
[17] Yang X, Harkins LK, Zubanova O,et al. Overexpression of Spry1 in chondrocytes causes attenuated FGFR ubiquitination and sustained ERK activation resulting in chondrodysplasia.Dev Biol. 2008;321(1):64-76.
[18] Shen L, Ling M, Li Y,et al. Feedback regulations of miR-21 and MAPKs via Pdcd4 and Spry1 are involved in arsenite-induced cell malignant transformation.PLoS One. 2013;8(3):e57652.
[19] Jin XL, Sun QS, Liu F,et al. microRNA 21-mediated suppression of Sprouty1 by Pokemon affects liver cancer cell growth and proliferation.J Cell Biochem. 2013;114(7): 1625-1633.
[20] Ling M, Li Y, Xu Y,et al. Regulation of miRNA-21 by reactive oxygen species-activated ERK/NF-κB in arsenite-induced cell transformation.Free Radic Biol Med. 2012;52(9):1508-1518.
[21] Urs S, Venkatesh D, Tang Y,et al. Sprouty1 is a critical regulatory switch of mesenchymal stem cell lineage allocation. FASEB J. 2010;24(9):3264-3273.
[22] Marson A, Levine SS, Cole MF,et al. Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells.Cell. 2008;134(3):521-533.
[23] Eloy-Trinquet S, Wang H, Edom-Vovard F,et al. Fgf signaling components are associated with muscles and tendons during limb development.Dev Dyn. 2009;238(5):1195-1206.
[24] Kaneto CM, Lima PS, Zanette DL,et al. COL1A1 and miR-29b show lower expression levels during osteoblast differentiation of bone marrow stromal cells from Osteogenesis Imperfecta patients.BMC Med Genet. 2014;15(1):45.
[25] Trohatou O, Zagoura D, Bitsika V,et al. Sox2 suppression by miR-21 governs human mesenchymal stem cell properties. Stem Cells Transl Med. 2014;3(1):54-68.
[26] Huszar JM, Payne CJ.MIR146A inhibits JMJD3 expression and osteogenic differentiation in human mesenchymal stem cells.FEBS Lett. 2014;588(9):1850-1856.
[27] 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.
[28] Kureel J, Dixit M, Tyagi AM,et al. miR-542-3p suppresses osteoblast cell proliferation and differentiation, targets BMP-7 signaling and inhibits bone formation.Cell Death Dis. 2014;5: e1050.
[29] Tiago DM, Marques CL, Roberto VP,et al. Mir-20a regulates in vitro mineralization and BMP signaling pathway by targeting BMP-2 transcript in fish.Arch Biochem Biophys. 2014;543: 23-30.
[30] Gong Y, Xu F, Zhang L,et al. MicroRNA expression signature for Satb2-induced osteogenic differentiation in bone marrow stromal cells.Mol Cell Biochem. 2014;387(1-2):227-239.
[31] Fakhry M, Hamade E, Badran B,et al. Molecular mechanisms of mesenchymal stem cell differentiation towards osteoblasts. World J Stem Cells. 2013;5(4):136-148.
[32] Jia J, Tian Q, Ling S,et al. miR-145 suppresses osteogenic differentiation by targeting Sp7.FEBS Lett. 2013;587(18): 3027-3031.
[33] Qureshi AT, Monroe WT, Dasa V,et al. miR-148b-nanoparticle conjugates for light mediated osteogenesis of human adipose stromal/stem cells.Biomaterials. 2013;34(31):7799-7810.
[34] Lee Y, Kim HJ, Park CK,et al. MicroRNA-124 regulates osteoclast differentiation.Bone. 2013;56(2):383-389.
[35] Baglìo SR, Devescovi V, Granchi D,et al. MicroRNA expression profiling of human bone marrow mesenchymal stem cells during osteogenic differentiation reveals Osterix regulation by miR-31.Gene. 2013;527(1):321-331.
[36] Gay I, Cavender A, Peto D,et al. Differentiation of human dental stem cells reveals a role for microRNA-218.J Periodontal Res. 2014;49(1):110-120.
[37] van Wijnen AJ, van de Peppel J, van Leeuwen JP,et al. MicroRNA functions in osteogenesis and dysfunctions in osteoporosis.Curr Osteoporos Rep. 2013;11(2):72-82.
[38] Ching ST, Cunha GR, Baskin LS,et al. Coordinated activity of Spry1 and Spry2 is required for normal development of the external genitalia.Dev Biol. 2014;386(1):1-11.
[39] Macià A, Vaquero M, Gou-Fàbregas M,et al. Sprouty1 induces a senescence-associated secretory phenotype by regulating NFκB activity: implications for tumorigenesis.Cell Death Differ. 2014;21(2):333-343.
[40] Purcell P, Jheon A, Vivero MP,et al. Spry1 and spry2 are essential for development of the temporomandibular joint.J Dent Res. 2012;91(4):387-393.