miR-20a介导流体剪切力促进细胞成骨分化的调控作用及机制

We had found that fluid shear stress (FSS) up-regulated the expression of miR-20a, and demonstrated that miR-20a mediated osteoblast differentiation induced by FSS. However, why miR-20a was up-regulated？ Further experiments showed that FSS phosphorylated Smads. A binding sequence with Smads in the miR-20a promoter core area was discovered by 5 'terminal sequence analysis. But which targets do miR-20a inhibited to promote osteoblast differentiation? The target genes of miR-20a included transduction negative regulator of BMP2 signal, such as BAMBI, Smad6, PPAR? and so on. Thus, we firstly proposed: FSS up-regulates miR-20a by Smads signal, miR-20a suppresses BAMBI, Smad6 or PPAR? and activates BMP2 signaling to promote osteoblast differentiation. This study is proposed to obtain reliable evidence that FSS up-regulates miR-20a by BMP2/Smads signal and miR-20a activates BMP2 signal, using the established experimental models and application of RNA interference, CALUX and CHIP technology. It is expected to demonstrate the function and mechanisms of miR-20a during osteoblast differentiation induced by FSS, so as to complement the new theory and regulatory targets for alveolar bone remodeling under mechanical stimulations.

我们已发现流体剪切力（FSS）上调miR-20a的表达，并证明miR-20a介导了FSS诱导的成骨细胞分化。miR-20a上调的机制是什么？证据表明FSS激活了Smads。5'末端序列续减分析确立了诱导miR-20a表达的启动子核心区并发现一个Smads结合序列。miR-20a抑制哪些靶点起作用？分析提示miR-20a很可能抑制BMP2信号转导负调控因子BAMBI、Smad6及PPAR?。由此，我们首次提出：FSS激活Smads上调miR-20a，miR-20a抑制BAMBI、Smad6或PPAR?，最终激活BMP2信号并促进成骨细胞分化。本研究拟通过前期实验模型，应用RNA干扰、CALUX及CHIP技术，旨在获得Smads直接上调miR-20a及miR-20a激活BMP2信号的可靠证据，阐明miR-20a对FSS促成骨细胞分化的作用和机制，从而为机械刺激促牙槽骨改建补充新的理论和调控靶点

项目的背景: 正畸牙移动是牙槽骨和牙周组织在机械力的作用下发现改建的一个过程。近年的研究表明，骨组织及骨细胞受到的力主要是流体剪切力（FSS），但FSS对成骨细胞的作用及其具体机制仍不明确。miRNA是一种单链非编码RNA，在基因转录后水平的调控中起重要的作用。最近的研究表明多种miRNAs参与了成骨分化的调控。但miRNA是否参与了FSS诱导的成骨分化仍不清楚。因此本研究旨在阐明在FSS诱导的成骨分化过程中miRNA的调控作用。.主要研究内容：本实验使用MC3T3-E1和BMSCs细胞作为研究对象。我们成功设计了平行平板流动装置及灌流系统对细胞加载模拟流体剪切力。随后采用基因芯片筛选FSS加载后差异表达的miRNA并进行PCR验证。采用miRNA过表达和表达抑制、荧光素酶报告基因及RNA干扰技术研究其在FSS促进成骨过程中具体作用机制。.重要结果和关键数据：FSS刺激MC3T3-E1和BMSCs细胞成骨分化过程中miR-20a/miR-19b/ miR-34a表达上调；过表达外源性miR-20a能促进成骨因子BMP2、RUNX2、SP7的表达，加速FSS诱导的MC3T3-E1细胞成骨分化，相反地，当敲低miR-20a后，BMP2、RUNX2、SP7的表达下调，抑制了FSS诱导的MC3T3-E1细胞成骨分化。双荧光素酶报告基因等方法证实BAMBI、SMAD6是miR-20a的靶基因，miR-20a分别抑制BMP2/Smads/RUNX2信号通路的负调控因子BAMBI、SMAD6，从而激活该通路，促进MC3T3-E1/BMSCs成骨细胞分化。.其科学意义：本实验证明了miR-20a通过抑制负调控因子BAMBI、SMAD6而激活BMP2/Smads/RUNX2信号通路，从而促进MC3T3-E1/BMSCs的成骨分化。该结果表明了正畸牙移动过程中miRNA参与了调控过程，有望成为治疗靶点，使正畸牙移动更加安全高效进行。