Post-transcriptional regulation of gene expression by microRNAs in antibody-mediated rejection.

: 具体目标。慢性抗体介导的排斥反应（AMR）是晚期慢性同种异体肾移植衰竭的主要原因。然而，尽管其临床重要性，但对移植肾如何调节对抗体和补体介导的攻击的反应的综合理解尚未建立。我们知识上的这一差距至少部分是由于对肾脏做出的导致基因调控变化的反应如何促进或预防损伤的不完全理解。 MicroRNA (miRNA) 是一类小型非编码 RNA，可在转录后调节基因表达。 miRNA 在调节肾损伤中发挥着重要作用。然而，AMR 中 miRNA 的研究很大程度上基于对疾病期间差异表达的总细胞 miRNA 的分析。这种方法是有问题的，因为它没有提供这些 miRNA 靶向的 mRNA 的信息。为了解决这个问题，我们通过分离与 RNA 结合蛋白 (RBP) AGO2 交联的 RNA，分离出 miRNA 及其在 RNA 诱导沉默复合物 (RISC) 中靶向的 mRNA。使用这种方法，我们定义了第一个移植相关肾损伤的 miRNA-mRNA 相互作用图。这些原理验证研究表明，在 miRNA-mRNA 靶组内，可以定义在遭受损伤的细胞中经历独特变化的 miRNA 及其靶向的 mRNA。通路富集分析表明，RISC 复合体中存在的 miRNA 靶向编码可能导致损伤的通路中的蛋白质的 mRNA。我们假设 miRNA-mRNA 靶组可用于识别导致 AMR 的基因途径。为了检验这一假设，我们将使用临床相关模型来确定 AMR 的 miRNA-mRNA 相互作用图或靶标组，并使用获得的靶向信息来检查 AMR 中 miRNA 调控下的基因通路的重要性。这些研究的一个关键问题是如何解释抗体介导的排斥（AMR）引起的表达变化的相关性。因此，在 R56 中，我们建议使用数字特殊表达谱来全局识别肾组织中表现出基因表达变化的区域，并将这种分析细化为不同的细胞类型并评估临床相关性。 具体目标 1：开发一种数字特殊分析方法来评估同种异体肾移植物中 miRNA 和 mRNA 表达的整体变化。在肾脏等异质组织中，确定 AMR 导致 miRNA 和 mRNA 表达发生变化的细胞类型至关重要。此外，定义变化发生的程度至关重要，以便优先考虑各种细胞类型的重要性。为了解决这个问题，我们建议在原理验证研究中使用 NanoString 的 GeoMx 数字空间分析器 (DSP) 进行数字特殊表达分析，以使用分析来全局识别正在经历 AMR 的小鼠肾组织中表现出基因表达变化的区域，并将这种分析细化为不同的细胞类型。我们将重点关注 miR-21，它在 AMR 期间的表达发生至少十倍的变化，并靶向已知的细胞死亡途径 mRNA。使用 DSP 与组织学相结合，我们将检查肾脏的哪些区域在 miR-21 及其靶标表达方面表现出类似的相对变化。一旦识别出表现出相似差异表达的区域，我们就可以使用标记的细胞类型特异性抗体和 RNA 探针进行额外的研究，以识别表现出变化的细胞类型，从而进一步完善我们的研究。 具体目标 2：在这个目标中，我们将通过检查移植患者活检材料中观察到的小鼠模型变化的程度来直接解决临床相关性。我们将使用如上所述的方法来确定在接受 AMR 的人类肾脏中是否观察到类似的变化。 叙事 慢性抗体介导的排斥反应（AMR）是晚期慢性同种异体肾移植衰竭的主要原因。然而，尽管其具有临床重要性，但对于移植肾如何调节对抗体和补体介导的攻击的反应尚未建立全面的理解。我们知识上的这一差距至少部分是由于对肾脏做出的导致基因调控变化的反应如何促进或预防损伤的不完全理解。 MicroRNA (miRNA) 是一类小型非编码 RNA，可在转录后调节基因表达。 miRNA 在调节肾损伤中发挥着重要作用。然而，AMR 中 miRNA 的研究很大程度上基于对疾病期间差异表达的总细胞 miRNA 的分析。这种方法是有问题的，因为它没有提供这些 miRNA 靶向的 mRNA 的信息。为了解决这个问题，我们通过分离与 RNA 结合蛋白 (RBP) AGO2 交联的 RNA，分离出 miRNA 及其在 RNA 诱导沉默复合物 (RISC) 中靶向的 mRNA。使用这种方法，我们定义了第一个移植相关肾损伤的 miRNA-mRNA 相互作用图。这些原理验证研究表明，在 miRNA-mRNA 靶组内，可以定义在遭受损伤的细胞中经历独特变化的 miRNA 及其靶向的 mRNA。通路富集分析表明，RISC 复合体中存在的 miRNA 靶向编码可能导致损伤的通路中的蛋白质的 mRNA。我们假设 miRNA-mRNA 靶组可用于识别导致 AMR 的基因途径。为了检验这一假设，我们将使用临床相关模型来确定 AMR 的 miRNA-mRNA 相互作用图或靶标组，并使用获得的靶向信息来检查 AMR 中 miRNA 调控下的基因通路的重要性。这些研究的一个关键问题是如何解释抗体介导的排斥（AMR）引起的表达变化的相关性。因此，在 R56 中，我们建议使用数字特殊表达谱来全局识别肾组织中表现出基因表达变化的区域，并将这种分析细化为不同的细胞类型。我们将使用这些信息来检查在作为 CTOT 网络的一部分收集的移植患者的活检材料中观察到这些变化的程度。

: Specific Aims. Chronic antibody-mediated rejection (AMR) is the major cause of late chronic renal allograft failure. Yet despite its clinical importance, an integrated understanding of how responses to antibody and complement mediated attack are regulated by the transplanted kidney has not been established. This gap in our knowledge is due at least in part to an incomplete understanding of how responses made by the kidney that result in changes in gene regulation promote or prevent injury. MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression post-transcriptionally. miRNAs play an important role in regulating renal injury. However, the study of miRNAs in AMR has largely been based on analysis of total cellular miRNAs that are differentially expressed during disease. This approach is problematic because it does not provide information about which mRNAs these miRNAs target. To address this issue, we isolated miRNAs and the mRNAs they are targeting in the RNA-Induced Silencing Complex (RISC) by isolating RNAs cross-linked to the RNA Binding Protein (RBP) AGO2. Using this approach we defined the first miRNA-mRNA interaction map for transplant related renal injury. These proof-of-principle studies revealed that within the miRNA-mRNA targetome it is possible to defined miRNAs and the mRNAs they target that undergo unique changes in cells undergoing injury. Pathway enrichment analysis indicated that miRNAs present in the RISC complex target mRNAs encoding proteins in pathways that may contribute to injury. We hypothesize that the miRNA-mRNA targetome can be used to identify gene pathways that contribute to AMR. To test this hypothesis, we will use a clinically relevant model to determine the miRNA-mRNA interaction map or targetome for AMR and use the targeting information obtained to examine the importance of gene pathways under regulation by miRNAs in AMR. A key issue for these studies is how to interpret the relevance of changes in expression due to antibody mediated rejection (AMR). In this R56 we therefore propose to use digital special expression profiling to globally identify areas in renal tissue that exhibit changes in gene expression and refine this analysis into distinct cell types and assess clinical relevance. Specific Aim 1: Developing a digital special profiling approach to assess global changes in miRNA and mRNA expression in renal allografts. In heterogeneous tissue such as the kidney it is critical to determine cell types exhibit alterations in miRNA and mRNA expression as a result of AMR. Moreover, it is critical to define the extent to which changes occur in order to prioritize the importance in various cell types. To address this issue, we propose to use digital special expression profiling using NanoString’s GeoMx Digital Spatial Profiler (DSP) in proof-of- principle studies to use profiling to globally identify areas in mouse renal tissue undergoing AMR that exhibit changes in gene expression and refine this analysis into distinct cell types. We will focus on miR-21, which undergoes at least a ten-fold change in expression during AMR and targets known cell death pathway mRNAs. Using DSP coupled with histology, we will examine which regions of the kidney exhibit similar relative changes in miR-21and expression of its targets. Once regions showing similar differential expression are identified, we can conduct additional studies using labeled cell-type specific antibodies and RNA probes to identify cell types exhibiting changes to further refine our studies. Specific Aim 2: In this aim we will directly address clinical relevance by examining the extent to which changes in our murine model are observed in biopsy material from transplant patients. We will use an approach like that described above to determine if similar changes are observed in human kidneys undergoing AMR. Narrative Chronic antibody-mediated rejection (AMR) is the major cause of late chronic renal allograft failure. Yet in spite of its clinical importance, an integrated understanding of how responses to antibody and complement mediated attack are regulated by the transplanted kidney has not been established. This gap in our knowledge is due at least in part to an incomplete understanding of how responses made by the kidney that result in changes in gene regulation promote or prevent injury. MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression post-transcriptionally. miRNAs play an important role in regulating renal injury. However, the study of miRNAs in AMR has largely been based on analysis of total cellular miRNAs that are differentially expressed during disease. This approach is problematic because it does not provide information about which mRNAs these miRNAs target. To address this issue, we isolated miRNAs and the mRNAs they are targeting in the RNA-Induced Silencing Complex (RISC) by isolating RNAs cross-linked to the RNA Binding Protein (RBP) AGO2. Using this approach we defined the first miRNA-mRNA interaction map for transplant related renal injury. These proof- of-principle studies revealed that within the miRNA-mRNA targetome it is possible to defined miRNAs and the mRNAs they target that undergo unique changes in cells undergoing injury. Pathway enrichment analysis indicated that miRNAs present in the RISC complex target mRNAs encoding proteins in pathways that may contribute to injury. We hypothesize that the miRNA- mRNA targetome can be used to identify gene pathways that contribute to AMR. To test this hypothesis, we will use a clinically relevant model to determine the miRNA-mRNA interaction map or targetome for AMR and use the targeting information obtained to examine the importance of gene pathways under regulation by miRNAs in AMR. A key issue for these studies is how to interpret the relevance of changes in expression due to antibody mediated rejection (AMR). In this R56 we therefore propose to use digital special expression profiling to globally identify areas in renal tissue that exhibit changes in gene expression and refine this analysis into distinct cell types. We will use this information to examine the extent to which these changes are observed in biopsy material from transplant patients that has been collected as part of the CTOT network.