组蛋白去甲基化酶KDM5C调控骨骼发育和骨代谢及其在骨折修复中的应用

KDM5C belongs to JARID subfamily of JmjC-containing proteins. The function of JmjC domain is to specifically demethylate di- and trimethylated lysine 4 on histone 3. The latest study found that mutations in the KDM5C could cause phenotypes such as short stature and poor mental development. However, there is a lack of in-depth understanding and analysis of its role in bone formation and bone metabolism, especially in adult bone reconstruction. The osteogenic differentiation of mesenchymal stem cells (MSCs) is the core process of bone formation. Our study showed that KDM5C silencing mediated by adeno-associated virus could significantly inhibit the osteogenic differentiation of MSCs. Then we constructed KDM5C gene knockout (KO) mice, and the knockout mice were significantly smaller than the wild control group. Based on the above analysis and combined with our experimental results, we speculated that "KDM5C can regulate one or some key genes of MSCs by histone methylation, which in turn affect the differentiation of MSCs and bone development of individuals, and transplantation of KDM5C overexpressing MSCs can promote fracture healing". We are planning to use KDM5C knockout mice and wild-type mice as the tool, through the RNA-seq, CHIP-seq, bone and cartilage staining, micro-CT techniques, as well as open femur fracture model of rats and mice, proving our hypothesis from gene-cell-individual level. This study will elucidate the mechanism of KDM5C in regulating bone development and bone metabolism, and provide new ideas and targets for stem cell therapy based on MSCs.

KDM5C属于JmjC结构域组蛋白去甲基化酶亚家族，介导了组蛋白H3K4me2/3去甲基化。最新研究发现KDM5C的突变会引起身材矮小、智力发育不良等表型。我们前期研究显示腺相关病毒介导的KDM5C沉默可以显著抑制MSCs的成骨分化，进而构建了KDM5C基因敲除小鼠，该基因敲除小鼠的体型明显小于野生对照组。基于上述并结合预实验结果，我们推测“KDM5C能够通过组蛋白甲基化修饰调节MSCs中的某个或某些关键基因，继而影响MSCs的分化和个体的骨骼发育，并且移植过表达KDM5C的MSCs能够促进骨折修复”。我们以KDM5C基因敲除小鼠和野生小鼠为工具，通过RNA-seq、CHIP-seq、骨-软骨整体染色和显微CT等技术，以及小鼠、大鼠的开放性股骨骨折模型，从基因-细胞-个体层次证明我们的假说。此研究将阐明KDM5C调控骨发育和骨代谢的机制，为以MSCs为基础的干细胞治疗提供新的思路和靶点。

KDM5C属于JmjC结构域组蛋白去甲基化酶亚家族，介导了组蛋白H3K4me2/3去甲基化。研究发现KDM5C的突变会引起身材矮小、智力发育不良等表型。我们的研究表明腺相关病毒介导的KDM5C沉默可以显著抑制MSCs的成骨分化。进而，我们构建了KDM5C基因敲除小鼠，骨-软骨整体染色和显微CT等结果表明该基因敲除小鼠的体型明显小于野生对照组，骨量明显降低，长骨变短等。通过提取KDM5C基因敲除小鼠和野生小鼠的MSCs进行比较发现，KDM5C敲除后MSCs成骨能力显著降低。我们进一步通过RNA-seq比较了野生型和KDM5C敲除MSCs的基因表达差异，KDM5C敲除MSCs中的HIF1α及wnt等信号通路被显著抑制，这些都进行了western blot和免疫组织化学染色分析验证。另一方面，在MSC中过表达KDM5C可以显著促进MSCs的成骨分化，并且HIF1α及wnt信号通路被激活，进一步提示KDM5C是通过这些信号通路调节MSCs的成骨分化，及骨骼发育。最后，通过开放性股骨骨折模型，我们证明了KDM5C过表达的MSCs可以有效促进骨折愈合。因此，本研究从基因-细胞-个体层次阐明了KDM5C调控骨发育和骨代谢的机制是通过影响HIF1α及wnt等信号通路，为以MSCs为基础的干细胞治疗提供新的思路和靶点。