p53/miR-34/WNT轴调控激素性股骨头坏死中成骨细胞凋亡的机制研究

Excessive osteoblast apoptosis induced by glucocorticoids is the primary pathologic manifestation of stroid-induced osteonecrosis of femoral head. Though activation of p53 function and inhibition of canonical Wnt signaling cascades are both documented in literature and our previous work, the links between these two pathways remain unclear. In order to prove miR-34 as the mediator of p53 and WNT, Firstly apoptosis of osteoblastic cell line MC3T3-E1 will be induced by dexamethasone treatment, and osteonecrosis tissue will be collected during operation, and then p53, microRNA-34 gene family(miR-34a/b/c) and WNT pathway-regulated genes will be tested in tissue and cell model; secondly siRNA will be adopted to silence p53 and then miR-34a/b/c and WNT pathway-regulated genes will be detected; subsequently miR-34a/b/c will be over-expressed and knockout , then WNT-related genes regulated by miR-34a/b/c will be investigated in silico and in vitro; ultimately mouse model of osteonecrosis will be induced by combination of dexamethason and endotoxin, in vivo miR-34a/b/c effect on osteoblast apoptosis and osteoncrosis will be illustrated. Through this project the relationship between p53 and WNT will be illustrated, after that the network regulating steroid-induced apoptosis of osteoblast and osteoncrosis will be constructed, then the mechanism of glucocorticoid induced osteonecrosis can be plan as a whole and the new target of therapy and diagnosis will be developed.

糖皮质激素诱导的成骨细胞凋亡是激素性骨坏死的主要病理表现，文献和我们的前期工作证实WNT通路受抑制和p53激活在凋亡启动中均发挥着重要的作用。为证明miR-34沟通p53和WNT形成调控网络，本项目首先复制成骨细胞凋亡模型，并收集激素性骨坏死标本，分别从细胞和组织水平检测过量激素对p53，miR-34家族（miR-34a/b/c）及WNT相关蛋白表达的影响；其次采用siRNA敲除p53后观察p53对miR-34a/b/c、WNT相关蛋白表达的调控作用；继而通过过表达和抑制miR-34a/b/c研究miR-34对p53及WNT的介导作用，结合荧光素酶分析验证其靶基因明确miR-34对WNT通路的调控作用；最后复制小鼠骨坏死模型，观察体内抑制miR-34a/b/c对骨坏死及成骨细胞凋亡的影响。通过研究，我们将整合p53和WNT通路,初步建立激素性骨坏死发病的调控网络，为开发新的诊治靶点奠定基础

因为发病机制不明，糖皮质激素诱导的股骨头坏死目前尚无确切有效的治疗方法。成骨细胞凋亡是激素性骨坏死的主要病理表现，前期研究证实WNT通路抑制和p53激活在成骨细胞凋亡启动中均发挥着重要作用，但二者的相互关系并不明确。为寻找沟通WNT通路抑制和p53激活的microRNA，我们首先检测了细胞水平和人体组织水平的p53、miR-34及WNT通路相关分子的活性，发现骨坏死区凋亡细胞比例增加，而WNT通路中WNT1、β-catenin和LEF表达降低，而P53和miR-34a增高；细胞水平也证实给予地塞米松刺激小鼠MC3T3-E1细胞后凋亡细胞比例增加，P53和miR-34a表达增加，而WNT1、β-catenin表达明显降低；我们进一步构建了P53siRNA质粒，敲除P53后地塞米松诱导的miR-34a上调得以逆转，而WNT1、β-catenin下调也得以逆转，提示P53是miR-34a和WNT通路的上游调控蛋白；但进一步敲低miR-34a时我们未能发现mirR-34a对地塞米松诱导的成骨细胞凋亡和增殖的逆转作用；为了进一步寻找P53是通过哪种microRNA调控WNT信号通路，我们通过Solexa测序技术比较了P53 敲低MC3T3-E1与野生型C3T3-E1细胞的microRNA表达谱，发现P53蛋白可以诱导包括miR-653-5p在内的295个上调、miR-129b-3p在内的316个下调microRNA，这些差异表达的microRNA主要参与铁死亡、细胞发育、干细胞多能性等方面。本研究发现miR-34a不是沟通P53和WNT调节凋亡关键节点分子，而P53直接调控的microRNA可作为下一步研究的关键分子，并为最终阐明糖皮质激素诱导的成骨细胞凋亡的分子机制奠定基础。