Multilineage regulation of mesenchymal stem cell differentiation by microRNAs

描述（由申请人提供）：间充质干/祖细胞（MSC）在广泛的组织再生治疗中具有相当大的潜力。虽然它们的分化能力已被广泛证明，但控制其可塑性的机制仍然知之甚少。我们实验室和其他实验室的观察表明，增殖的MSC在诱导分化之前表达谱系相关分子。这导致了一种建议的分化模型，其中向特定细胞类型的定型是由沿诱导途径增加的表达沿着和与其他谱系相关的基因的抑制的组合效应引起的。microRNA是内源性表达的小RNA，可以调节这一过程。通过作为转录抑制因子，它们最近成为癌症和干细胞中细胞分化的调节因子。在这个提议中，我们将研究特定microRNA及其基因靶点对MSC分化的作用。我们对经历成骨和软骨分化的MSC进行了microRNA分析，并将miR-130 b、miR-432和miR-559鉴定为两种谱系中差异表达的miRNA。MSC的功能研究表明，miR-130 b的多谱系调节;其过表达增强成骨细胞表型和抑制平滑肌分化。在miR-130 b转染的MSC中的差异基因表达分析已经确定了几个mRNA靶点，这些靶点可以作为成骨和成肌分化的控制点。基于这些发现，本提案的目标1将确定miR-130 b的直接靶点，并评估其在骨、软骨和肌源性分化中的作用。将遵循相同的方法来评估miR-432和-559的功能效应和基因靶点。在具体目标2中，我们将研究这些microRNA的功能效应是否在体内复制。将通过将编码模拟物和发夹抑制剂的慢病毒载体系统性递送至发育中的小鼠胚胎来进行MicroRNA过表达和抑制实验。在特定的发育阶段，将评价胚胎组织的骨、软骨和平滑肌形成。这项研究的成功完成将1）确定间充质细胞中miRNA介导的基因沉默如何改变骨-软骨-和肌源性谱系的承诺，2）确定这些特定microRNA在肌肉骨骼发育过程中的潜在调控作用。这将提高我们目前对间充质干细胞分化的理解，并有助于未来基于细胞的肌肉骨骼修复疗法。 公共卫生相关性：干细胞/祖细胞移植可促进损伤或疾病后的骨和软骨修复。成功的修复需要通过分化过程从植入的干细胞产生稳定的骨/软骨细胞。本项目将研究小调控RNA（microRNA）在控制干细胞向特定细胞类型分化途径中的作用。这将揭示新的机制，可用于治疗软骨缺损修复和骨折愈合。

DESCRIPTION (provided by applicant): Mesenchymal stem/progenitor cells (MSCs) hold considerable potential for a wide range of tissue regeneration therapies. While their differentiation capacity has been demonstrated extensively, mechanisms that control their plasticity remain poorly understood. Observations by our laboratories and others have shown that proliferating MSCs express lineage-associated molecules prior to induction of differentiation. This has led to a suggested model of differentiation where commitment to a specific cell type results from the combined effects of increased expression along the induced pathway and repression of genes related to other lineages. MicroRNAs are endogenously expressed, small RNAs that may regulate this process. By acting as transcriptional repressors, they have recently emerged as regulators of cellular differentiation in cancer and stem cells. In this proposal we will investigate the role of specific microRNAs and their gene targets on MSC differentiation. We have performed microRNA profiling on MSCs undergoing osteogenic and chondrogenic differentiation and identified miR-130b, miR-432 and miR-559 as differentially expressed miRNAs among both lineages. Functional studies in MSCs suggest multilineage regulation by miR-130b; its overexpression enhanced the osteoblast phenotype and repressed smooth muscle differentiation. Differential gene expression analysis in miR-130b transfected MSCs has identified several mRNA targets that could serve as control points for osteo- and myogenic differentiation. Building on these findings, Aim 1 of this proposal will identify direct targets of miR-130b and assess their role in osteo-, chondro- and myogenic differentiation. The same approach will be followed to assess the functional effects and gene targets of miR-432 and -559. In Specific Aim 2, we will investigate whether the functional effects of these microRNAs are replicated in vivo. MicroRNA overexpression and inhibition experiments will be performed by systemic delivery of lentiviral vectors encoding mimics and hairpin inhibitors to developing mouse embryos. At specific developmental stages embryonic tissues will be evaluated for bone, cartilage and smooth muscle formation. Successful completion of this study will 1) determine how miRNA-mediated gene silencing in mesenchymal cells alters commitment to osteo- chondro- and myogenic lineages and 2) identify potential regulatory roles for these specific microRNAs during musculoskeletal development. This should improve our current understanding of mesenchymal stem cell differentiation and aid future cell-based therapies for musculoskeletal repair. PUBLIC HEALTH RELEVANCE: Bone and cartilage repair, following injury or disease, may be facilitated by the transplantation of stem/progenitor cells. Successful repair requires the generation of stable bone/cartilage cells from implanted stem cells through the process of differentiation. This project will study the roles of small regulatory RNAs (microRNAs) in controlling the differentiation pathways of stem cells towards specific cell types. This should uncover novel mechanisms that can be exploited for therapeutic use in cartilage defect repair and fracture healing.