miR-195 effect on osteogenic differentiation of human bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2016.45.001

Abstract

Abstract:

BACKGROUND: Previous studies have found that the expression level of miR-195 in differentiated human bone marrow mesenchymal stem cells (hBMMSCs) is significantly higher than that in undifferentiated hBMMSCs. However, miR-195 effect during this differentiation process and possible mechanism remain unclear.
OBJECTIVE: To explore the effect of miR-195 on osteogenic differentiation of hBMMSCs and possible mechanism.
METHODS: hBMMSCs were isolated and cultured in vitro. Alkaline phosphatase activity, Runx2, osteopontin and SMAD7 protein expression and miR-195 expression level during osteogenic differentiation of hBMMSCs were determined by alkaline phosphatase kit, western blot and real-time PCR, respectively. miR-195-downexpressed hBMMSCs were constructed by lipofection transfection, and were used to investigate the effect of miR-195 was on osteogenic differentiation of hBMMSCs. Dual luciferase reporter assay was used to identify whether the 3’UTR of SMAD7 mRNA was a binding target of miR-195. In addition, we transfected hBMMSCs with SMAD7 cDNA (pcDNA-SMAD7), and investigated the effect of SMAD7 on osteogenic differentiation of hBMMSCs.
RESULTS AND CONCLUSION: The isolated and cultured hBMMSCs had good osteogenic differentiation ability in vitro. Expression level of miR-195 was increased with the increasing of induction time, and the expression level of SMAD7 was reversed. miR-195 promoted osteogenic differentiation of hBMMSCs. Luciferase assay confirmed that miR-195 targeted SMAD7 directly, and overexpression of SMAD7 inhibited the osteogenic differentiation of hBMMSCs. Taken together, miR-195 promotes osteogenic differentiation of hBMMSCs by targeting SMAD7.

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 Development, Mesenchymal Stem Cells, MicroRNAs, Tissue Engineering

CLC Number:

R394.2

Song Peng, Jing Kai, Xue Jian-hua, Wei Peng-fei. miR-195 effect on osteogenic differentiation of human bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(45): 6693-6699.

Figures/Tables 1

2.1 人骨髓间充质干细胞成骨分化能力通过检测碱性磷酸酶活性以及成骨分化特异基因Runx2和osteopontin表达水平来观察人骨髓间充质干细胞成骨分化能力，发现碱性磷酸酶活性以及Runx2和osteopontin的表达水平均随着诱导时间的增加而升高，但在第9天和12天各项指标的水平没有明显差异(图1)。这些结果表明分离培养的人骨髓间充质干细胞具有优良的体外成骨分化能力。 2.2 人骨髓间充质干细胞成骨分化miR-195和SMAD7表达通过qRT-PCR和免疫印迹分别检测了miR-195以及它的一个潜在靶SMAD7在诱导过程中的表达水平变化，发现miR-195随诱导时间的增加而升高，SMAD7则相反，同样地，它们在第9天和12天的表达水平也无明显差异(图2)。 2.3 miR-195促进人骨髓间充质干细胞成骨分化为了探究miR-195对人骨髓间充质干细胞向成骨细胞分化影响，首先通过脂质体转染构建miR-195低表达的细胞和对照细胞，然后诱导其分化，在分化的第9天，检测碱性磷酸酶活性，发现miR-195低表达组相比于对照组，碱性磷酸酶活性明显降低(图3A)。此外，采用免疫印迹检测两组细胞中骨分化标志物Runx2和osteopontin蛋白的表达。结果显示miR-195低表达组相比于对照组有明显较低的Runx2和osteopontin蛋白表达水平(图3B)。这些结果表明了miR-195能够促进人骨髓间充质干细胞向成骨细胞的分化进程。 2.4 miR-195直接靶向SMAD7 通过Targetscan和miRanda等在线软件预测miR-195靶基因，发现Smad7是它的一个潜在靶，SMAD7 mRNA的3’UTR与miR-195的种子区存在理论上的互补碱基配对序列(图4A)。进而执行了荧光素酶实验，结果显示共转染SMAD7野生型的实验组的荧光素酶活性明显低于对照组，而共转染SMAD7突变型的实验组与对照组的荧光素酶活性无显著差异，这证实miR-195可以直接作用于SMAD7(图4B)。又进一步实验，研究转染miR-195抑制剂和抑制剂对照对人骨髓间充质干细胞中SMAD7蛋白表达量的影响。结果显示转染miR-195抑制剂组相比于对照组有明显较高的SMAD7蛋白表达水平(图4C)。此外，又进一步探究miR-195是否通过靶向SMAD7促进人骨髓间充质干细胞向成骨细胞分化，首先通过脂质体转染构建SMAD7高表达的细胞(pcDNA-SMAD7)和对照细胞(pcDNA-control)，然后诱导其分化，在分化的第9天，检测碱性磷酸酶活性，发现SMAD7高表达组的碱性磷酸酶活性明显低于对照组(图4D)。免疫印迹实验显示SMAD7高表达组相比于对照组有明显较低的Runx2和osteopontin蛋白表达水平(图4E)。以上结果表明，miR-195通过靶向SMAD7促进人骨髓间充质干细胞成骨分化。"

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