Dysregulated microRNA function in diamond blackfan anemia

钻石黑扇贫血症中 microRNA 功能失调

• 批准号: 8439603
• 负责人: CARL D NOVINA
• 金额: $ 34.77万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-29 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8439603
• 关键词: Affect; Anabolism; Anemia; Aspirate substance; BFU-E; Biological; Biopsy; Bone Marrow; Bone Marrow Cells; Bone Tissue; CFU-E; Candidate Disease Gene; Cartilage; Cells; Complementary DNA; Data; Data Set; Defect; Development; Diamond; Diamond-Blackfan anemia; Equilibrium; Erythroblasts; Erythroid; Erythropoiesis; Functional RNA; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Mutation; Gene Targeting; Genes; Genetic Translation; Grant; Hair; Hematopoiesis; Hematopoietic stem cells; Hour; Human; In Vitro; Individual; Inherited; Link; Malignant Neoplasms; Maps; Mediating; Messenger RNA; MicroRNAs; Molecular; Molecular Profiling; Mutation; Normal tissue morphology; Oncogenes; Outcome; Pancytopenia; Pathway Analysis; Pathway interactions; Patients; Phenotype; Population; Protein Biosynthesis; RNA; RNA Interference; Resolution; Ribosomal Proteins; Ribosomes; Risk; Role; Sampling; Site; Small Interfering RNA; Source; Stress; Syndrome; Technology; Testing; Tissue Sample; Translational Repression; Translations; base; cancer risk; clinical phenotype; genome-wide; high throughput screening; induced pluripotent stem cell; insight; mRNA Expression; mRNA Stability; novel; progenitor; public health relevance; rapid growth; reconstitution; ribosomal protein 3; stem; transcriptome sequencing; tumor

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.

描述（由申请人提供）：Diamond Blackfan Anemia （DBA）是一种遗传性骨髓衰竭综合征，以红细胞生成减少和癌症风险增加为特征。核糖体蛋白基因（rpg）的先天性突变与该综合征最密切相关。目前尚不清楚为什么RPG表达的减少会选择性地减少红细胞生成和易患癌症，而癌症通常需要增加快速生长所需的蛋白质合成。我们最近的数据可能提供了在DBA中观察到的RPG表达减少和临床表型之间的分子联系。通过高通量筛选发现人类microRNA （miRNA）功能的影响因子和调节因子，发现rpg是一类调节miRNA活性的新基因。具体来说，每个RPG的敲低降低了miRNA活性，从而增加了miRNA靶mrna的表达。在本应用中，我们同样提出，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的表达（两者都富集于miRNA靶点），导致红细胞生成减少，增加了癌症风险。为了测试DBA红细胞生成减少的机制，我们将获得DBA患者和正常供体的骨髓和组织样本。我们将从这两种来源产生诱导多能干细胞（iPS）。我们将比较正常供者的骨髓和iPS与DBA患者的骨髓和iPS在RPG基因敲低和未敲低情况下的红细胞生成情况。我们将描述和分离这些来源的离散红系祖细胞。然后，我们将从每个祖细胞中分离总RNA、单体RNA和多体RNA，并进行miRNA （Luminex头）和mRNA （RNA-seq）表达谱分析。转录组和翻译组图谱将生成并相互比较，并与匹配对照样本的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，以确定当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，以期在RPG敲低和DBA患者样本中逆转红细胞生成表型。我们将使用sirna或特定的miRNA模拟物来敲除那些mRNA水平或翻译在RPG表达减少时显著增加的基因。相反，当RPG表达减少时，我们将使用cdna来重组总mRNA水平或翻译量明显下降的基因。我们的研究将建立RPG表达、miRNA功能、miRNA靶向基因表达和红细胞生成之间的新联系，并可能揭示DBA治疗的新靶点。