Role of microRNAs in regulation of the marrow microenvironment

描述（由申请人提供）：造血的调节是复杂的，涉及几种不同的细胞类型及其基因产物在骨髓微环境（ME）中共同发挥作用;目前对这些基因的协调表达如何调节的了解甚少。通过微小RNA（miR）和其他非编码RNA调节基因表达已被用来解释大型脊椎动物如何具有高度特化的细胞和组织功能，尽管具有有限数量的蛋白质编码转录本。我们假设miR以重要的方式促进造血调节，并形成反式作用调节因子的功能网络的一部分。从2个功能不同的基质细胞系的分析产生的初步数据揭示了几个miR的差异表达，这些miR可能靶向促成其不同表型的因子的转录物。例如，这些细胞系之一命名为HS 5，其表达的miR-886- 3 p比第二种命名为HS 27 a的细胞系多40倍。发现这种miR直接下调CXCL 12的表达，CXCL 12是一种已知对造血干细胞（HSC）归巢至其小生境至关重要的趋化因子。虽然可以从这些常规方法中获得重要的信息来定义miR-mRNA相互作用（表达谱分析、生物信息学预测算法和基于过表达或敲低的功能研究），但它们未能证明体内直接的miR-mRNA相互作用。因此，我们优化了最近描述的生物化学技术，该技术鉴定了在RISC（RNA诱导沉默复合物）背景下相互作用的那些miR和mRNA。该技术被称为HITS-CLIP（交联免疫沉淀后的高通量测序），其利用紫外（UV）辐射将miR和mRNA交联至蛋白Argonaute，Argonaute是RISC的组分，随后是其免疫沉淀和RNA的高通量测序。本申请提出了一种基于HITS-CLIP的系统生物学方法来定义miR在造血ME中的作用。在目的1中，我们将定义miR-886- 3 p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。在目的2中，我们将利用HITS-CLIP方法系统地确定ME的4个不同细胞群体中的miR-mRNA相互作用：两种基质细胞类型（定义为HS 5和HS 27 a），巨噬细胞和内皮细胞。将鉴定并功能验证靶向对维持造血干细胞（HSC）小生境至关重要的3个基因（CXCL 12、血管生成素1和锯齿蛋白1）的特异性miR。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命运决定的基因组的特定功能网络。结果将提供一个更好的理解造血ME是如何调节的，这是至关重要的设计新的治疗血液系统疾病的ME失调。

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.