Fracture Repair in Aging: Identifying networks by miRNA and mRNA co-sequencing

临床上迫切需要开发新的骨再生疗法来治疗延迟愈合和骨不连 骨折和严重的骨缺损。此外，老化导致骨骼修复严重受损。一个关键 骨折愈合的一个方面始于骨膜祖细胞的扩张，其立即被激活 然后分化成骨形成成骨细胞和软骨细胞。然而，分子 控制它们响应损伤的活化、扩增和分化的机制仍然有限。 microRNA（miRNA）介导的基因表达的转录后调控在肿瘤的发生发展中起着关键作用。 干细胞向成骨细胞或软骨细胞谱系的定向和分化。由于单个 miRNA可以潜在地靶向数百种不同的mRNA，识别控制基因表达的网络。 在我们对骨骼生物学和修复的理解中，miRNAs仍然代表着一个重大的差距。 在年轻骨和老年骨之间可以观察到miRNA表达的差异，并且可能介导一些 老化对骨折修复的影响。 无偏倚的全转录组分析揭示了基因表达的重要信息 活跃在静止的骨膜和愈合骨折的程序。研究利用从整个 骨已经记录了这些组织中mRNA或miRNA的变化。然而，单细胞RNA 测序研究很容易证明细胞异质性存在于骨膜和再生中， 骨，使得在这些复杂组织中大量RNAseq研究的解释不太清楚。的能力 观察骨折修复过程中单个细胞中协调的mRNA-miRNA网络将提供范例- 转移信息，可以用来加强修复。此外，基于miRNA的治疗剂的局部递送 代表了一种促进老年人骨愈合的令人兴奋的新策略。的透彻理解 在愈合骨中的每种细胞类型中活跃的mRNA-miRNA网络将提供关键的理论基础， miRNAs在治疗环境中的靶向作用。 我们建议对分离的非造血谱系细胞进行单细胞mRNA-miRNA共测序， 从静止骨膜和骨折激活的骨膜，比较年轻和老年小鼠。这将允许 我们确定基于表达的细胞亚群和mRNA-miRNA相互作用网络， 人群，以及在骨折修复和衰老过程中这些是如何调节的。用于单细胞miRNA的方案- mRNA共测序是最近才开发的。骨折修复的复杂微环境是 这是这项新技术的理想应用，它将填补我们对骨骼生物学理解的一个重要空白 并为进一步研究基于miRNA的骨修复疗法提供了理论基础。

There is an urgent clinical need to develop new bone regenerative therapeutics to treat delayed and non-union fractures, and critical sized bone defects. Moreover, aging results in severely impaired skeletal repair. A critical aspect of fracture healing begins with expansion of periosteal progenitors, which are activated immediately after injury and then differentiate into bone forming osteoblasts and chondrocytes. However, the molecular mechanisms controlling their activation, expansion, and differentiation in response to injury remains limited. microRNA (miRNA) mediated post-transcriptional regulation of gene expression plays a key role in the commitment and differentiation of stem cells toward the osteoblastic or chondrogenic lineages. Since a single miRNA can potentially target hundreds of distinct mRNAs, identifying the gene expression networks controlled by miRNAs continues to represent a significant gap in our understanding of skeletal biology and repair. Differences in miRNA expression can be observed between young and old bone, and likely mediate some of the impact of aging on fracture repair. Non-biased whole transcriptome analyses have revealed important information about gene expression programs active in the resting periosteum and in healing fractures. Studies utilizing RNA extracted from whole bone have documented changes in either mRNA or miRNAs in these tissues. However, single cell RNA sequencing studies readily demonstrate the cellular heterogeneity present in periosteum and in regenerating bone, making less clear the interpretation of bulk RNAseq studies in these complex tissues. The ability to observe coordinated mRNA-miRNA networks in individual cells during fracture repair will provide paradigm- shifting information that could be used to enhance repair. Further, local delivery of miRNA-based therapeutics represents an exciting novel strategy for facilitating bone healing in the elderly. A thorough understanding of the mRNA-miRNA networks active in each cell type in the healing bone will provide critical rationale for miRNAs to target in a therapeutic setting. We propose to perform single cell mRNA-miRNA co-sequencing on non-hematopoietic lineage cells isolated from resting periosteum and periosteum activated by fracture, comparing young and aged mice. This will allow us to identify expression-based cell subpopulations and mRNA-miRNA interaction networks within those populations, and how these are modulated during fracture repair and in aging. Protocols for single cell miRNA- mRNA co-sequencing were only recently developed. The complex micro-environment of fracture repair is an ideal application for this new technology, which will fill a significant void in our understanding of skeletal biology and provide a rationale for further investigation of miRNA-based therapeutics for bone repair.