miR-21/Sprouty1 function axis regulates the osteogenic differentiation of bone marrow mesenchymal stem cells after postmenopausal osteoporosis

doi:10.3969/j.issn.2095-4344.2017.21.002

Abstract

Abstract:

BACKGROUND: Osteogenic differentiation is a complex process involving transcriptional and post-transcriptional regulation by multiple signaling pathways, and the specific mechanisms remain unclear. It is of great significance to study the role of critical miRNAs in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in the treatment of osteoporosis and bone defects.
OBJECTIVE: To explore the regulatory ability of miR-21/Sprouty1 function axis in the osteogenic differentiation of BMSCs in patients with postmenopausal osteoporosis.
METHODS: BMSCs were isolated from healthy people (H-hBMSCs) and patients with postmenopausal osteoporosis (PMOP-hBMSCs), and their osteogenic ability was compared. Expression of miR-21 and Spry1 at gene and protein levels was detected by real-time RT-PCR and western blot assay, respectively. miR-21 expression was upregulated via transfection in PMOP-hBMSCs, and the osteogenic ability and Spry1 expression of the cells were detected, while real-time RT-PCR and western blot were used to detect the expression of osteogenic marker genes, Runx2 and Osterix.
RESULTS AND CONCLUSION: Compared with H-hBMSCs, PMOP-hBMSCs osteogenic ability was weakened significantly, miR-21 expression decreased, and Spry1 expression increased, indicating an inhibition to the miR-21-Spry1 function axis. Through the transfection of miR-21 and down-regulation of Spry1, the expression levels of Runx2 and Osterix were increased, and PMOP-hBMSCs osteogenic ability was partially restored.

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

Key words: Bone Marrow, Mesenchymal Stem Cells, MicroRNAs, Osteoporosis, Postmenopausal, Tissue Engineering

CLC Number:

R394.2

Yang Nan, Zhou Wei, Wang Guang, Ding Yin, Jin Yan. miR-21/Sprouty1 function axis regulates the osteogenic differentiation of bone marrow mesenchymal stem cells after postmenopausal osteoporosis[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(21): 3287-3292.

Figures/Tables 1

2.1 干细胞培养和鉴定结果对照组和绝经后骨质疏松组患者的骨髓间充质干细胞多为梭形，呈成纤维细胞样，很少一部分为大的扁平状。流式细胞仪检测细胞表面标记，发现高表达间充质干细胞表面标志物STRO-1和CD146，同时也表达间充质标志物CD29，CD90，CD44。 2.2 绝经后骨质疏松患者骨髓间充质干细胞的成骨分化能力减弱通过real time RT-PCR和Western Blot检测成骨分化过程中特异性表达基因Runx2和Osterix的表达，结果发现Runx2和Osterix在绝经后骨质疏松患者骨髓间充质干细胞中的表达低于正常人骨髓间充质干细胞。这一结果表明绝经后骨质疏松患者骨髓间充质干细胞成骨分化能力减弱(图1)。 2.3 miR-21在绝经后骨质疏松患者骨髓间充质干细胞中的表达量下降为了进一步证明绝经后骨质疏松患者骨髓间充质干细胞成骨能力的减弱是否与miR-21表达和功能异常有关，实验检测了原代培养的绝经后骨质疏松患者骨髓间充质干细胞和正常人骨髓间充质干细胞中miR-21的表达。Real time RT-PCR结果显示，miR-21在绝经后骨质疏松患者骨髓间充质干细胞中的表达明显低于正常人骨髓间充质干细胞(图2)。 2.4 Spry1在绝经后骨质疏松患者骨髓间充质干细胞中表达明显上升为了验证绝经后骨质疏松患者骨髓间充质干细胞成骨能力的减弱是否与Spry1的异常表达相关，实验检测了原代培养的绝经后骨质疏松患者骨髓间充质干细胞和正常人骨髓间充质干细胞中Spry1的表达。Real time RT PCR和 Western Blot结果显示，Spry1在绝经后骨质疏松患者骨髓间充质干细胞中的表达明显高于正常人骨髓间充质干细胞。这一结果与miR-21表达下降正好相反，说明miR-21-Spry1功能轴的异常表达可能和绝经后骨质疏松患者骨髓间充质干细胞成骨能力减弱有关(图3)。 2.5 高表达miR-21能够恢复绝经后骨质疏松患者骨髓间充质干细胞受损的成骨能力为了验证miR-21表达量下降是否和绝经后骨质疏松患者骨髓间充质干细胞成骨分化能力减弱相关，绝经后骨质疏松患者骨髓间充质干细胞转染miR-21的前体(pre-miR-21)和前体对照(pre-miR阴性对照)，正常人骨髓间充质干细胞转染前体对照，转染2 d后进行成骨诱导，分别于第7天和第21天进行碱性磷酸酶染色和茜素红染色比较3组细胞的成骨能力。结果显示，转染率为80%，高表达miR-21的绝经后骨质疏松患者骨髓间充质干细胞的碱性磷酸酶活性和钙结节的染色面积高于转染前体对照组，但是略低于正常人骨髓间充质干细胞转染前体对照组(图4 A-C)。此外，采用real time RT-PCR和Western Blot分析成骨特异性基因Runx2和Osterix mRNA和蛋白表达。结果发现，成骨诱导7 d后，高表达miR-21组Runx2和Osterix的mRNA水平和蛋白水平明显高于转染前体对照组，但是略低于正常对照组(图4D，E)。同时还检测了3组细胞中Spry1的mRNA和蛋白表达水平。结果显示，与转染前体对照组相比，高表达miR-21组中Spry1的mRNA和蛋白表达明显下降，但是仍略高于正常对照组(图4F，G)。"

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