miRNA双靶向基因调控PPARγ和GREM 1预防激素性股骨头坏死的实验研究

Steroid-induced osteonecrosis of femoral head(ONFH) is a common disease that can lead to physical disability in high rate. The effective preventive means are poor at present. The novel pathogenesis of the ONFH have indicated that steroid can up-regulate expression of adipogenic gene PPARγ and down-regulate expression of osteogenic gene in the bone marrow mesenchymal stem cells (BMSCs), and then increase adipogenesis and reduce osteogenesis of the cells. We have demonstrated that siRNA can prevent steroid-induced the adipogenic differentiation and maintained osteogenic differentiation in BMSCs, which play a preventive role. It has been known, a single microRNA (miRNA) can regulate and control multiple target genes which is better effect than siRNA. In order to explore the higher efficient preventive measures with double effect that could inhibit the adipogenesis and promote the osteogenesis of the cell simultaneously, the project aims to adopt the miRNA dual targeting gene regulating, utilizing gene chip technology to screen the low expression miRNAs in the necrotic tissue of steroid-induced ONFH model of animal, through bioinformatics analysis to select the objective miRNA simultaneously targeting adipogenic PPARγ gene and GREM 1 gene. GREM 1 is antagonist of osteogenic gene BMP-2 gene. The objective miRNA is transfected to rat BMSCs in vitro. The recombinant lentivirus vector for expressing objective miRNA is constructed and injected into the animal femoral head to transfect the cell in vivo. The increasing objective miRNA may suppress the expression of PPARγ and GREM 1 in the cells and up-regulating expression of BMP-2, which can efficiently inhibit adipogenic differentiation and proactively promote osteogenic differentiation of the cells, and then more efficiently prevent the onset of steroid-induced ONFH from the key aspects. This new idea has a more important scientific significance and application prospects.

激素性股骨头坏死是致残率高的常见病，缺乏有效预防。重要发病机制是激素上调BMSCs内成脂基因PPARγ表达，下调成骨基因表达，细胞成脂增多且成骨减少。我们证明siRNA能阻止激素诱导细胞成脂分化，维持其成骨分化，起到预防作用。已知一个miRNA可调控多个靶基因，优于siRNA。为了探索既能阻止细胞成脂又能促进成骨的双倍高效预防方法，本项目拟采用miRNA双靶向基因调控，利用基因芯片技术筛选激素性股骨头坏死组织中的低表达miRNAs，通过生物信息学分析筛选出同时靶向成脂基因PPARγ与成骨基因BMP-2拮抗剂GREM 1的目标miRNA，转染大鼠体外BMSCs，阻断细胞PPARγ与GREM 1表达，上调BMP-2表达，高效阻止细胞成脂分化，主动促进成骨分化，为更高效地预防激素性ONFH提供实验依据，这一新思路具有更加重要的科学意义和应用前景。

骨髓间充质干细胞（BMSCs）的成脂分化与成骨分化在激素性股骨头坏死（ONFH）的发病机制中起着重要的作用。已有研究表明某些microRNAs（miRNAs）可参与调控细胞的成脂与成骨分化。在本研究中，我们首先建立了一个大鼠激素性ONFH模型，通过基因芯片检测和生物信息学分析在模型中筛选出9个表达明显上调和28个表达明显下调的miRNAs。应用TargetScan和miRBase进行生物信息学分析，筛选出同时以过氧化物酶体增殖物激活受体-γ（PPARγ）及Gremlin-1(GREM1，BMP-2的拮抗剂）为靶基因的目标miRNA（miR-27a）。本研究结果显示，miR-27a表达与PPARγ及GREM 1表达呈负相关性。通过双荧光素酶报告基因检测证实PPARγ和GREM1均为miR-27a的靶基因。细胞学实验采用油红O染色、ALP染色、ELISA、荧光定量PCR、Western-blotting等方法检测miR-27a干预激素诱导大鼠BMSCs分化的分子生物学特性，结果证实，给予激素大鼠的BMSCs转染miRNA-27a，miR-27a表达上调，可显著下调细胞内PPARγ、GREM1表达，并上调成骨基因Runx2、BMP-2表达，有效抑制激素诱导的BMSCs成脂分化，并促进细胞成骨分化。且与单一沉默PPARγ或GREM1实验对照比较证明，上调miRNA-27a表达对激素诱导的BMSCs成脂分化的抑制及促进成骨分化的作用更为显著。. 总之，本研究通过基因芯片检测和生物信息学分析确定了包括miR-27a在内的37个与激素性ONFH密切相关差异表达的miRNAs，miR-27a直接靶向作用于PPARγ和GREM1，可有效阻止BMSCs成脂分化并促进其成骨分化。表明miRNA-27a是阻止激素诱导BMSCs成脂分化的一个关键调控因子，可作为防治ONFH的潜在靶点，这为进一步深入研究防治激素性ONFH开创了新的科研思路，具有重要科学意义和研究应用前景。