Dysregulated microRNA function in diamond blackfan anemia

描述（由申请人提供）：Diamond Blackfan贫血（DBA）是一种遗传性骨髓衰竭综合征，其特征是红细胞生成减少和癌症风险增加。核糖体蛋白基因（RPGs）的先天性突变与该综合征关系最为密切。目前还不清楚为什么减少RPG表达选择性地减少红细胞生成和易患癌症，这些癌症通常需要增加快速生长所必需的蛋白质合成。我们最近的数据可能提供了减少RPG表达和DBA中观察到的临床表型之间的分子联系。一项旨在发现人类microRNA（miRNA）功能的效应子和调节子的高通量筛选将RPGs鉴定为一类调节miRNA活性的新型基因。具体地说，每个RPG的敲低降低了miRNA活性，从而增加了miRNA靶mRNA的表达。在本申请中，我们类似地提出，在DBA中，RPG突变降低了miRNA活性，从而改变了发育调节基因和癌基因（均富含miRNA靶位点）的表达，导致红细胞生成减少和癌症风险增加。为了测试DBA中红细胞生成减少的这种机制，我们将获得DBA患者和正常供体的骨髓和组织样品。我们将从这两种来源产生诱导多能干细胞（iPS）。我们将比较来自正常供体的具有和不具有RPG敲低的骨髓和iPS的红细胞生成与来自DBA患者的骨髓和iPS。我们将从这些来源中的每一个中表征和分离离散的红系祖细胞。然后，我们将从每个祖细胞中分离总RNA、单体RNA和多聚体RNA，并进行miRNA（Luminex珠）和mRNA（RNA-seq）表达谱分析。将生成转录组和翻译组图谱，并相互比较，并与匹配对照样品的miRNA表达谱进行比较。我们将使用这些数据集作为无偏基因网络分析的输入，以确定RPG表达减少时受影响的（miRNA调节的）途径。然后，我们将通过操纵基因的表达来确认基因在预测网络中的作用，以期逆转RPG敲除和DBA患者样本中的红细胞生成表型。我们将使用siRNA或特定的miRNA模拟物来敲除当RPG表达降低时mRNA水平或翻译强烈增加的基因。相反，我们将使用cDNA来重建当RPG表达降低时总mRNA水平或翻译强烈降低的基因。我们的研究将在RPG表达、miRNA功能、miRNA靶向基因表达和红细胞生成之间建立新的联系，并可能揭示DBA治疗的新靶点。

DESCRIPTION (provided by applicant): Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome characterized by reduced erythropoiesis and increased cancer risk. Congential mutations in ribosomal protein genes (RPGs) are most closely linked with this syndrome. It is unclear why decreased RPG expression selectively decreases erythropoiesis and predisposes to cancers that typically require increased protein synthesis necessary for rapid growth. Our recent data may provide a molecular connection between reduced RPG expression and the clinical phenotypes observed in DBA. A high-throughput screen to discover effectors and regulators of human microRNA (miRNA) function identified RPGs as a novel class of genes regulating miRNA activity. Specifically, knockdown of every RPG decreased miRNA activity and thus increased expression of miRNA-target mRNAs. In this application, we similarly propose that in DBA, RPG mutations reduce miRNA activity which alters expression of developmentally-regulated genes and oncogenes (both enriched in miRNA target sites), leading to reduced erythropoiesis and increased cancer risk. To test this mechanism of decreased erythropoiesis in DBA, we will obtain DBA patient and normal donor bone marrow and tissue samples. We will generate induced pluripotent stem (iPS) cells from both sources. We will compare erythropoiesis from bone marrows and iPS from normal donors with and without RPG knockdown to bone marrows and iPS from DBA patients. We will characterize and isolate discreet erythroid progenitors from each of these sources. We will then isolate total RNA, monosomal RNA, and polysomal RNA from each progenitor and perform miRNA (Luminex bead) and mRNA (RNA-seq) expression profiling. Transcriptome and translatome maps will be generated and compared to each other and to miRNA expression profiles from matched control samples. We will use these data sets as inputs for unbiased gene network analyses to identify affected (miRNA-regulated) pathways when RPG expression is reduced. We will then confirm the roles of genes in the predicted networks by manipulating their expression in hopes of reversing erythropoiesis phenotypes in RPG knockdown and DBA patient samples. We will use siRNAs or specific miRNA mimics to knockdown genes whose mRNA levels or translation robustly increased when RPG expression was reduced. Conversely, we will use cDNAs to reconstitute genes whose total mRNA levels or translation robustly decreased when RPG expression was reduced. Our studies will establish novel connections between RPG expression, miRNA function, miRNA-targeted gene expression, and erythropoiesis, and may reveal new targets for DBA therapy.