Bmal1与miRNA的交互作用对骨衰老的影响及其机制

The modulating mechanism of the aging of bone marrow-derived mesenchymal stem cells（BMSCs）and bone is still unclear. .Our previous study illustrated that core circadian clock gene Bmal1, which would bind to miR-142-3p and miR-155-5p, played a crucial role in aging, and Hippo pathway was closely related to the senescence of BMSCs. So we hypothesize that there are BMSCs specific-expressed miRNAs regulating Bmal1, which may occur crosstalk between them, and lead to BMSCs aging via Hippo pathway..To verify this hypothesis, ChIP-Seq, database searching and computer algorithm to predict was applied to screen and identify the Bmal1-target miRNA which associated with bone senescence and circadian rhythm. Then miRNA, Bmal1, Hippo pathway will be controlled respectively by CRISPR/Cas9, gene transfection, signaling pathway regulation and other advanced techniques to test the interaction among them and the changes of aging and osteogenesis related factors in downstream. Stem cell gene therapy will also be applied to Bmal1bmsc-/-/aging mouse to verify if Bmal1 and miRNA can reverse bone aging in vivo. Accordingly, we hope to further elucidate the molecular mechanism of circadian clock gene regulating bone aging, and provide new targets for prevention and treatment of bone aging, which may bring new views on stem cell therapy.

骨衰老的调控机制尚不清楚。课题组前期发现：Bmal1调控着BMSCs的衰老，且可能与miR-142-3p、miR-155-5p等存在相互作用，并与Hippo通路活动密切相关。由此提出：BMSCs中存在特异性miRNA，调控着Bmal1表达，二者还存在交互作用，通过Hippo通路，引起成骨及衰老相关变化，导致骨衰老。为验证本假说，课题组前期以ChIP-Seq、文库检索、软件预测、体外验证，锁定目标miRNA，并通过CRISPR/Cas9、基因转染、信号通路调控等手段，分别控制Bmal1、目标miRNA、Hippo通路，检测它们之间的相互作用及衰老、成骨因子的联动效应，进而对Bmal1条件性敲除/自然衰老小鼠行颅颌/股骨局部干细胞基因注射，体内验证Bmal1、miRNA延缓／逆转骨衰老的作用，以期阐明Bmal1调控骨衰老的分子机制，为防治骨衰老提供新靶点，为干细胞治疗提供新思路。

骨衰老的调控机制尚不清楚。课题组前期发现:Bmal1调控着BMSCs的衰老，且可能与miR- 142-3p、miR-155-5p等存在相互作用，并与Hippo通路活动密切相关。由此提出: BMSCs中存在特异性miRNA，调控着Bmal1表达，二者还存在交互作用，通过Hippo通路，引起成骨及衰老相关变化，导致骨衰老。为验证本假说，课题组以ChIP-Seq、文库检索、软件预测、体外验证等方式，证实目标miRNA确为miR142-3p/miR155-5p，进而通过基因转染、信号通路调控等手段，分别控制Bmal1 、miR142-3p/miR155-5p、Hippo通路，检测得出它们之间存在crosstalk，调控其中的任一部分均可产生衰老、成骨因子的联动效应，进而对Bmal1条件性敲除/自然衰老小鼠行颅颌/股骨局部干细胞基因注射，体内验证了Bmal1、miR142-3p/miR155-5p逆转骨衰老的作用，为阐明Bmal1调控骨衰老的分子机制、防治骨衰老提供了新的靶点，为干细胞治疗提供了新思路。