miR-20a-5p contributes to osteogenic differentiation of human dental pulp stem cells by regulating BAMBI and activating the phosphorylation of Smad5 and p38.
miR-20a-5p contributes to osteogenic differentiation of human dental pulp stem cells by regulating BAMBI and activating the phosphorylation of Smad5 and p38.
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miR-20a-5p通过调节BAMBI并激活Smad5和p38的磷酸化促进人牙髓干细胞的成骨分化
DOI：
10.1186/s13287-021-02501-8
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发表时间：
2021-07-22
影响因子：
7.5
通讯作者：
Zhao Z
中科院分区：
文献类型：
--
作者：
Cen X;Pan X;Zhang B;Huang W;Pei F;Luo T;Huang X;Liu J;Zhao Z
背景人牙髓干细胞（human dental pulp stem cells，hDPSCs）是颅颌面骨组织再生的首选种子细胞。作为miR-17-92簇的成员，miR-20 a-5 p在骨重建过程中起着重要的调节作用。本研究旨在探讨miR-20 a-5 p在hDPSCs成骨过程中的作用及其机制。方法采用定量逆转录-聚合酶链反应（qRT-PCR）方法检测miR-20 a-5 p在hDPSCs成骨过程中的表达。我们通过干扰miR-20 a-5 p在hDPSCs中的表达来阐明miR-20 a-5 p在体外和体内成骨中的功能。双荧光素酶报告基因检测证实miR-20 a-5 p与BAMBI之间存在直接结合位点，并通过细胞共转染研究miR-20 a-5 p的作用机制。抑制miR-20 a-5 p可减弱ALP/ARS染色强度，下调成骨标志物mRNA和蛋白表达;过表达miR-20 a-5 p可增强ALP/ARS染色强度，上调成骨标志物表达。micro-CT重建图像和组织学结果均显示miR-20 a-5 p可促进颅骨缺损的再生。miR-20 a-5 p直接靶向骨形态发生蛋白和激活素膜结合抑制剂（BAMBI），后者是hDPSC成骨的抑制剂。沉默BAMBI部分逆转了miR-20 a-5 p敲低对骨生成的抑制作用。miR-20 a-5 p沉默后，Smad 5和p38磷酸化水平降低，而miR-20 a-5 p过表达或BAMBI沉默后，p-Smad 5和p-p38磷酸化水平上调。
BackgroundHuman dental pulp stem cells (hDPSCs) are the preferable choice of seed cells for craniomaxillofacial bone tissue regeneration. As a member of the miR-17-92 cluster, miR-20a-5p functions as an important regulator during bone remodeling. This study aimed to investigate the roles and mechanisms of miR-20a-5p during osteogenesis of hDPSCs.MethodsQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to determine the expression of miR-20a-5p during osteogenesis of hDPSCs. We interfered with the expression of miR-20a-5p in hDPSCs to clarify the function of miR-20a-5p on osteogenesis both in vitro and vivo. Direct bind sites between miR-20a-5p and BAMBI were confirmed by dual-luciferase reporter assay, and the underlying mechanisms were investigated with cell co-transfections.ResultsThe expression of miR-20a-5p was showed to be upregulated during osteogenesis of hDPSCs. Inhibition of miR-20a-5p could weaken the intensity of ALP/ARS staining and downregulate the expression of mRNAs and proteins of osteogenic markers, while overexpression of miR-20a-5p could enhance the intensity of ALP/ARS staining and the expression of osteogenic markers. Both micro-CT reconstruction images and histological results showed that miR-20a-5p could promote the regeneration of calvarial defects. miR-20a-5p directly targeted bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), and the latter one was an inhibitor of hDPSC osteogenesis. Silencing BAMBI partially reversed the suppression effect of miR-20a-5p knockdown on osteogenesis. Phosphorylation of Smad5 and p38 was decreased when miR-20a-5p was silenced, whereas p-Smad5 and p-p38 were upregulated when miR-20a-5p was overexpressed or BAMBI was silenced.ConclusionsIt is demonstrated that miR-20a-5p functioned as a regulator of BAMBI to activate the phosphorylation of Smad5 and p38 during osteogenic differentiation of hDPSCs.
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通讯作者：
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