Role of microRNAs in regulation of the marrow microenvironment

描述(由申请人提供)：造血的调控是复杂的，涉及几种不同类型的细胞及其基因产物在骨髓微环境(ME)的背景下共同发挥作用；目前对这些基因的协调表达如何调控知之甚少。通过microRNAs(MiRs)和其他非编码RNA对基因表达的调节已经被用来解释大型脊椎动物如何在蛋白质编码转录本数量有限的情况下具有高度专业化的细胞和组织功能。我们假设MIR在造血调控中起重要作用，并形成反式作用调节的功能网络的一部分。对2个功能不同的基质细胞系的初步分析数据显示，几个miR的差异表达可能针对导致其不同表型的因子的转录本。例如，其中一个被命名为HS5的细胞系表达的miR-886-3p是另一个被称为HS27a的细胞系的40倍。这种miR被发现直接下调CXCL12的表达，CXCL12是一种已知对造血干细胞(HSC)归巢至其利基至关重要的趋化因子。虽然这些定义miR-mRNA相互作用的传统方法(表达谱、生物信息预测算法和基于过度表达或敲除的功能研究)可以获得重要信息，但它们不能证明在体内直接的miR-mRNA相互作用。因此，我们优化了最近描述的一种生化技术，该技术可以识别在RISC(RNA诱导沉默复合体)中相互作用的miR和mRNAs。这项技术被称为HITS-CLIP(高通量测序遵循交链免疫沉淀)，利用紫外线(UV)将miRs和mRNAs与蛋白质ArgAerte交联，ArgAerte是RISC的一个组成部分，然后进行免疫沉淀和高通量RNA测序。本申请提出了一种基于HITS-CLIP的系统生物学方法来定义miRs在造血ME中的作用。在目标1中，我们将定义miR-886-3p的小鼠同源物，并建立一个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ME的四个不同细胞群体中miR-mRNA的相互作用：两种间质细胞类型(由HS5和HS27a定义)、巨噬细胞和内皮细胞。针对3个对维持造血干细胞(HSC)生态位至关重要的基因(CXCL12、Angiopoietin1和Jagged1)的特定MIR将被识别和功能验证。在目标3中，我们将使用系统生物学的方法来组织生成的HITS-CLIP数据，以预测和测试特定的功能网络，这些网络调节可能指导HSC命运决定的基因组。这一结果将使我们更好地了解造血ME是如何被调节的，这对于设计ME调节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.