Role of microRNAs in regulation of the marrow microenvironment

microRNA在调节骨髓微环境中的作用

• 批准号: 8255482
• 负责人: Manoj M. Pillai
• 金额: $ 36.08万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-08 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8255482
• 关键词: Algorithms; Binding; Biochemical; Bioinformatics; Biological Assay; Blood Cells; Bone Marrow; CXCL12 gene; Cell Line; Cell Maintenance; Cell surface; Cells; Coculture Techniques; Code; Complex; Data; Data Set; Development; Disease; Endothelial Cells; Endothelium; Functional RNA; Gene Expression; Gene Expression Regulation; Genes; Goals; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Homing; Homologous Gene; Human; Immune; Immunoprecipitation; Ligands; Maintenance; Maps; Marrow; Measures; Messenger RNA; Methodology; MicroRNAs; Molecular Profiling; Mus; Pathway Analysis; Phenotype; Play; Population; Precipitation; Production; Proteins; RNA Sequences; RNA-Induced Silencing Complex; Regulation; Role; Stromal Cells; Systems Biology; Techniques; Testing; Tissues; To specify; Trans-Activators; Transcript; Transplantation; Ultraviolet Rays; Vertebrates; Work; Xenograft Model; base; cell type; chemokine; crosslink; falls; hematopoietic stem cell fate; implantation; in vivo; in vivo Model; innovation; knock-down; mRNA Transcript Degradation; macrophage; novel; novel therapeutics; prevent; public health relevance; receptor; stem cell niche; stem cell population; transcription factor

DESCRIPTION (provided by applicant): Regulation of hematopoiesis is complex, involving several different cell types and their gene products functioning together in the context of the marrow microenvironment (ME); there is little understanding currently of how the coordinated expression of these genes is regulated. Regulation of gene expression by microRNAs (miRs) and other non-coding RNAs has been invoked to explain how large vertebrates have highly specialized cellular and tissue functions despite having limited number of protein coding transcripts. We hypothesize that miRs contribute in a significant way to hematopoietic regulation and form part of a functional network of trans- acting regulators. Preliminary data generated from the analysis of 2 functionally distinct stromal cell lines reveal differential expression of several miRs that may target transcripts of factors which contribute to their distinct phenotypes. For example, one of these cell lines designated HS5 expresses >40 fold more miR-886-3p than a second one, called HS27a. This miR was found to directly down-regulate the expression of CXCL12, a chemokine known to be critical to the homing of the hematopoietic stem cell (HSC) to its niche. Although important information can be gained from such conventional approaches to defining miR-mRNA interactions (expression profiling, bio-informatic prediction algorithms and functional studies based on over-expression or knock-down), they fall short of demonstrating direct miR-mRNA interactions in vivo. Hence we have optimized a recently described biochemical technique that identifies those miRs and mRNAs interacting with each other in the context of the RISC (RNA-Induced Silencing Complex). This technique, termed HITS-CLIP (High Through-put Sequencing following Cross-Linked Immuno Precipitation) utilizes ultraviolet (UV) radiation to cross-link miRs and mRNAs to the protein Argonaute, a component of the RISC followed by its immune precipitation and high throughput sequencing of the RNA. This application proposes a systems-biology approach based on HITS-CLIP to define the role of miRs in the hematopoietic ME. In Aim 1, we will define the murine homologues of miR-886-3p and develop a novel in vivo model to test the function of these miRs in the ME. In Aim 2, we will utilize the HITS-CLIP methodology to systematically define the miR-mRNA interactions in 4 distinct cellular populations of the ME: two stromal cell types (as defined by HS5 and HS27a), macrophages and endothelial cells. Specific miRs that target 3 genes critical to maintaining the hematopoietic stem cell (HSC) niche (CXCL12, Angiopoietin1 and Jagged1) will be identified and functionally validated. In Aim 3, we will use a systems-biology approach to organize the HITS-CLIP data generated to predict and test specific functional networks that regulate groups of genes that may direct the HSC fate decisions. The results will provide a better understanding of how the hematopoietic ME is regulated, which is critical to devising new therapies for hematologic disorders where the ME is dysregulated. PUBLIC HEALTH RELEVANCE: The goal of this project is to define the mechanisms by which different cells in the bone marrow microenvironment interact to regulate blood cell production while retaining a stem cell population. Defining these mechanisms will inform the development of better therapies for diverse blood disorders.

描述（由申请人提供）：造血的调节很复杂，涉及几种不同的细胞类型及其基因产物在骨髓微环境的背景下一起起作用（ME）；目前，几乎没有了解这些基因的协调表达。尽管蛋白质编码的转录本数量有限，但已调用MicroRNA（MIR）和其他非编码RNA对基因表达的调节，以解释大型脊椎动物具有高度专业的细胞和组织功能。我们假设MIR可以为造血调节做出重要贡献，并成为跨性调节剂功能网络的一部分。从对2个功能上不同的基质细胞系的分析产生的初步数据揭示了几种miR的差异表达，这些miR可能针对因素的转录本，这些因素有助于其独特的表型。例如，这些指定HS5的细胞系之一表达> 40倍的miR-886-3p，比第二个称为HS27A的细胞系更高。发现该miR直接下调了CXCL12的表达，CXCL12是一种趋化因子，这对于将造血干细胞（HSC）归为至关重要。尽管可以从这种常规方法中获得重要的信息来定义miR-mRNA相互作用（表达分析，基于过表达或敲低的表达算法和功能研究），但它们却没有证明体内直接的miR-mRNA相互作用。因此，我们优化了一种最近描述的生化技术，该技术在RISC的背景下（RNA诱导的沉默复合物）识别了这些miR和mRNA相互作用。该技术称为旋转式旋转（交联免疫沉淀后的高贯穿测序），利用紫外线（UV）辐射来交联的miR和mRNA和蛋白质Argonaute，蛋白质Argonaute是RISC的组成部分，RISC的组成部分是其免疫沉淀和RNA的高吞吐量测序。该应用提出了一种基于HITS-CLIP的系统生物学方法，以定义MIR在造血ME中的作用。在AIM 1中，我们将定义miR-886-3p的鼠同源物，并开发出一种新颖的体内模型来测试这些miR在ME中的功能。在AIM 2中，我们将利用hits-clip方法系统地定义了ME的4个不同细胞种群中的miR-MRNA相互作用：两种基质细胞类型（由HS5和HS27A定义），巨噬细胞和内皮细胞。靶向3个基因对维持造血干细胞（HSC）小众（CXCL12，Angiopoietin1和Jagged1）至关重要的特定miR将被鉴定并在功能上验证。在AIM 3中，我们将使用一种系统生物学方法来组织生成的HITS-CLIP数据，以预测和测试特定的功能网络，以调节可能指导HSC命运决策的基因组。结果将更好地理解造血ME的调节方式，这对于为ME失调的血液学疾病设计新疗法至关重要。 公共卫生相关性：该项目的目的是定义骨髓微环境中不同细胞相互作用以调节血细胞产生同时保留干细胞群体的机制。定义这些机制将为各种血液疾病的更好疗法的发展提供信息。