Role of microRNAs in regulation of the marrow microenvironment

描述（由申请人提供）：造血调节是复杂的，涉及几种不同的细胞类型及其基因产物在骨髓微环境（ME）的背景下共同起作用；目前对这些基因的协调表达是如何被调控的了解甚少。微小rna和其他非编码rna对基因表达的调控被用来解释大型脊椎动物如何在蛋白质编码转录物数量有限的情况下具有高度特化的细胞和组织功能。我们假设miRs在造血调节中起着重要的作用，并构成了反式作用调节因子的功能网络的一部分。对2种功能不同的基质细胞系的分析产生的初步数据揭示了几种miRs的差异表达，这些miRs可能针对导致其不同表型的因子的转录本。例如，其中一种称为HS5的细胞系表达的miR-886-3p比另一种称为HS27a的细胞系表达的miR-886-3p多40倍。该miR被发现直接下调CXCL12的表达，CXCL12是一种趋化因子，已知对造血干细胞（HSC）归巢到其生态位至关重要。尽管从这些定义miR-mRNA相互作用的常规方法（表达谱、生物信息学预测算法和基于过表达或敲除的功能研究）中可以获得重要信息，但它们无法在体内证明miR-mRNA的直接相互作用。因此，我们优化了最近描述的生化技术，该技术可以识别在RISC （rna诱导沉默复合体）背景下相互作用的miRs和mrna。这项技术被称为HITS-CLIP（高通量测序后交联免疫沉淀）利用紫外线（UV）辐射将miRs和mrna交联到Argonaute蛋白上，Argonaute蛋白是RISC的一个组成部分，随后是其免疫沉淀和RNA的高通量测序。本应用提出了一种基于HITS-CLIP的系统生物学方法来定义mir在造血ME中的作用。在Aim 1中，我们将定义miR-886-3p的小鼠同源物，并开发一种新的体内模型来测试这些mir在ME中的功能。在目标2中，我们将利用HITS-CLIP方法系统地定义miR-mRNA在4种不同的ME细胞群中的相互作用：两种基质细胞类型（由HS5和HS27a定义），巨噬细胞和内皮细胞。针对维持造血干细胞（HSC）生态位至关重要的3个基因（CXCL12， Angiopoietin1和Jagged1）的特异性miRs将被鉴定和功能验证。在目标3中，我们将使用系统生物学方法来组织生成的HITS-CLIP数据，以预测和测试调节可能指导HSC命运决定的基因群的特定功能网络。该结果将更好地理解造血ME是如何被调节的，这对于设计针对ME失调的血液疾病的新疗法至关重要。

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.