miR-106a regulates the osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting bone morphogenetic protein receptor 2

doi:10.3969/j.issn.2095-4344.2016.28.001

Abstract

Abstract:

BACKGROUND: Under normal physiological conditions, there is a homeostasis between the osteogenic and adipogenic differentiation of human bone marrow mesenchymal stem cells. Osteogenic differentiation is an important process in the formation of skeleton in which differentiated and mature osteoblasts are indispensable.
OBJECTIVE: To explore the role of miR-106a and its target gene, bone morphogenetic protein receptor 2 (BMPR2) in the differentiation of human bone marrow mesenchymal stem cells into osteoblasts.
METHODS: Human bone marrow mesenchymal stem cells were induced to differentiate into osteoblasts by osteoblast-specific induction medium, and this process was detected by western blot and alkaline phosphatase staining. Overexpression of miR-106a was elicited by transfecting miR-106a mimics and the BMPR2 knockdown achieved by RNA interference. The expression levels of miR-106a and BMPR2 were detected by real-time PCR and western blot, respectively. The interaction of miR-106a and BMPR2 was verified by TargetScan software and dual luciferase report experiment assay.
RESULTS AND CONCLUSION: The expression of miR-106a was decreased whereas the expression of BMPR2 increased with the progress of osteogenesis differentiation. When miR-106a was overexpressed, alkaline phosphatase activity was declined and the expressions of runt-related transcription factor 2 and osteocalcin, markers of osteogenesis differentiation, both decreased. The expression of BMPR2 was decreased as well. BMPR2 was predicted to be the target gene of miR-106a by TargetScan software and this prediction proved by dual luciferase report experiments assay. Additionally, osteogenesis differentiation was inhibited by knocking down the expression of BMPR2. These results indicate that miR-106a regulates the differentiation of bone marrow mesenchymal stem cells into osteoblasts by targeting BMPR2.

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: MicroRNAs, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Gu Su. miR-106a regulates the osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting bone morphogenetic protein receptor 2[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(28): 4109-4116.

Figures/Tables 2

2.1 人骨髓间充质干细胞成功分化为成骨细胞 Runx2和OCN是成骨分化的标志蛋白，在成骨分化过程中，碱性磷酸酶活性上升，Runx2和OCN表达上升[27]。通过检测细胞Runx2和OCN蛋白的表达水平和碱性磷酸酶活性可以判断间充质干细胞成骨分化的进程。作者将人骨髓间充质干细胞使用成骨诱导培养基诱导分化，试剂盒检测碱性磷酸酶活性，western blot检测Runx2，OCN及骨形态发生受体蛋白2表达，real-time PCR检测分化前后miR-106a表达。结果表明，随着分化诱导时间的增加，碱性磷酸酶活性逐渐上升，成骨分化标志蛋白Runx2和OCN的表达也不断上升，说明在实验过程中人骨髓间充质干细胞被成功诱导为成骨细胞。随着成骨分化的进行，miR-106a表达逐渐下降，暗示miR-106a在成骨分化过程中有负调控作用。骨形态发生受体蛋白2表达逐渐升高，在第8天达到高峰，和miR-106a的变化趋势相反(图1)。这些结果暗示在骨髓间充质干细胞成骨分化的过程中，miR-106a和骨形态发生受体蛋白2的表达可能存在负相关性。 2.2 miR-106a抑制骨髓间充质干细胞向成骨细胞分化为了探索miR-106a在成骨分化过程中的作用，作者将miR-106a模拟剂转染至骨髓间充质干细胞，检测转染miR-106a模拟剂对分化指标的影响。结果表明转染miR-106a模拟剂后，miR-106a表达量上升，碱性磷酸酶活性下降，成骨分化标志蛋白Runx2和OCN表达量下降(图2)，表明miR-106a模拟剂抑制了骨髓间充质干细胞向成骨细胞的分化。 2.3 miR-106a靶向骨形态发生受体蛋白2抑制骨髓间充质干细胞向成骨细胞分化使用Target Scan软件在线查找miR-106a的靶基因，结果发现，骨形态发生受体蛋白2是miR-106a的潜在靶基因。图3A是Target Scan预测的骨形态发生受体蛋白2和miR-106a的绑定位点。为了检测miR-106a能否调控骨形态发生受体蛋白2表达，作者使用Western blot检测了转染miR-106a模拟剂后骨形态发生受体蛋白2蛋白表达情况。结果发现转染miR-106a模拟剂后，骨形态发生受体蛋白2蛋白表达下降(图3B)。使用荧光素酶报告系统，共转染miR-106a和骨形态发生受体蛋白2 3’-UTR野生型或骨形态发生受体蛋白23’-UTR突变型，转染36 h后收集细胞测定荧光素酶活性。结果发现，和转染miR-106a阴性对照和骨形态发生受体蛋白2 3’-UTR野生型的细胞相比，共转染miR-106a和骨形态发生受体蛋白2 3’-UTR野生型的细胞荧光素酶活性显著下降，miR-106a模拟剂对骨形态发生受体蛋白2 3’-UTR突变型荧光素酶活性没有抑制作用。这些结果表明miR-106a可以抑制骨形态发生受体蛋白2 3’-UTR的转录活性，从而调控骨形态发生受体蛋白2的表达(图3)。 2.4 敲降骨形态发生受体蛋白2抑制骨髓间充质干细胞向成骨细胞分化为了研究骨形态发生受体蛋白2在骨髓间充质干细胞成骨分化中的作用，作者使用RNA干扰技术敲降骨形态发生受体蛋白2，诱导成骨分化并检测成骨分化相关指标。如图4A所示，转染骨形态发生受体蛋白2 siRNA的细胞骨形态发生受体蛋白2表达量下降了80%，干扰效果较好。敲降骨形态发生受体蛋白2表达和过表达miR-106a的结果类似，碱性磷酸酶活性下降(图4B)，成骨分化标志蛋白Runx2和OCN的表达下降(图4C)。这些结果表明敲降骨形态发生受体蛋白2表达，人骨髓间充质干细胞成骨分化受到抑制。"

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