Defining LncRNA Function in normal and Shwachman Diamond Syndrome Myelopoiesis
Defining LncRNA Function in normal and Shwachman Diamond Syndrome Myelopoiesis
批准号:
10320386
负责人:
CARL D NOVINA
金额:
$44.13万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2019
资助国家:
美国
项目状态:
已结题
起止时间:
2019-01-15 至 2023-12-31
关键词:
AffectAffinityBindingBinding ProteinsBinding SitesBioinformaticsBiological AssayBiological MarkersBiologyBone MarrowBone marrow failureCD34 geneCell DeathCell physiologyCellsChildClinical TrialsColorectal NeoplasmsDNADataDefectDevelopmentDiagnosisDiseaseDisease ProgressionEctopic ExpressionEpitopesExocrine pancreasFailureFunctional disorderFutureGenesGenetic DiseasesGoalsHOXA10 geneHeartHematologyHematopoiesisHematopoieticHematopoietic Stem Cell TransplantationHematopoietic stem cellsHybridsImmune systemIndividualInflammationInheritedLibrariesLiverMALAT1 geneManuscriptsMarrowMessenger RNAMethodsMolecularMorbidity - disease rateMultipotent Stem CellsMyelogenousMyelopoiesisOutcomeOxidative StressPathogenesisPathogenicityPathway interactionsPatientsPhysiologyPlayProcessProteinsRNARNA SequencesRNA-Binding ProteinsRNA-Protein InteractionRegulationRoleSequence AnalysisShwachman-Diamond syndromeSignal TransductionStructureSystemTestingTimeTissuesTransplantationUntranslated RNAYeastsbasebioinformatics pipelinebody systembonebone marrow failure syndromeclinical developmentcurative treatmentsdifferential expressionimproved outcomeinhibitorinsightknock-downleukemiamortalitynovelpre-clinicalprogenitorprotein functionprotein protein interactionself-renewalsingle-cell RNA sequencingtranscriptome sequencingvirtual
中文摘要
项目摘要
Shwachman-Diamond综合征是一种累及多个器官的遗传性骨髓衰竭综合征
系统。然而,SDS中的血液学缺陷仍然缺乏特征性。我们使用的是单细胞RNA
序列测定以确定转化生长因子信号在造血干细胞(HSCs)和
多能祖细胞(MPP),但不是血统承诺的祖细胞。我们还发现显著减少了
正常情况下下调转化生长因子信号的lncRNA的表达。我们的数据表明在SDS骨骼中存在IncRNAs
骨髓衰竭。
为了明确lncRNAs如何在正常的骨髓生成中发挥作用,以及它们的失调是如何在抑郁综合征中致病的,我们
将使用最近开发的酵母三杂交(Y3H)试验来系统地定义lncRNA-蛋白质
在正常和疾病造血中发现的100个lncRNA的相互作用。Y3H能够探测到低-
丰度和低亲和力相互作用在核糖核酸-蛋白质相互作用中。覆盖lncRNA-蛋白质相互作用
现有的PPI网络将定义lncRNA调节的正常骨髓生成途径及其
抑郁状态的调节失调。因为我们将筛选100个单独的lncRNA的重叠lncRNA片段,
这些研究将使系统的lncRNA结构-功能分析成为可能。通过这一途径获得的信息
结构-功能分析对于建立序列和/或结构参数至关重要,序列和/或结构参数可能
能够预测lncRNA与蛋白质的结合活性,从而促进我们对LncRNA-蛋白质结合活性的生物学理解
LncRNA,甚至是未包括在此初始屏幕中的那些。
然后,我们将验证lncRNA在造血和改变转化生长因子激活中的作用。具体地说,我们将使用
CD34细胞缺失SBDS并增加(通过异位表达)或减少(通过敲除)lncRNAs
以及它们相互作用的蛋白,并测试这些在自我更新、髓系承诺、增殖、
细胞死亡、氧化应激、炎症和转化生长因子激活。要进一步测试特定LncRNAs在
在转化生长因子信号转导过程中,我们还将检测转化生长因子抑制剂挽救正常造血功能的能力。我们会
在受影响的亚群中确定转化生长因子反应基因,这些基因可以作为新的抑郁疗法的靶点;以及
检测其在十二烷基硫酸钠造血中的作用。因此,这些研究将(1)鉴定lncRNA、蛋白质和lncRNA-
蛋白质相互作用指导正常和SDS致病髓系功能;(2)开发一个平台
系统地研究lncRNAs;和(3)提供临床前证据,以支持临床试验靶向
LncRNA指导的未来抑郁治疗的途径。
英文摘要
Project Summary
Shwachman-Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome affecting multiple organ
systems. However, the hematological defects in SDS remain poorly characterized. We used single cell RNA
sequencing to identify increased TGF signaling specifically in hematopoietic stem cells (HSCs) and
multipotent progenitors (MPPs), but not lineage-committed progenitors. We also identified significantly reduced
expression of lncRNAs that normally down-regulate TGF signaling. Our data implicate lncRNAs in SDS bone
marrow failure.
To define how lncRNAs function in normal myelopoiesis and how their dysregulation is pathogenic in SDS, we
will use a recently developed yeast three hybrid (Y3H) assay to systematically define lncRNA-protein
interactions for 100 lncRNAs identified in normal and disease hematopoiesis. Y3H is capable of detecting low-
abundance and low-affinity interactions lncRNA-protein interactions. Overlaying the lncRNA-protein interacting
onto existing PPI networks will define lncRNA-regulated pathways of normal myelopoiesis and their
dysregulation in SDS. Because we will screen of overlapping lncRNA fragments of 100 individual lncRNAs,
these studies will enable systematic lncRNA structure-function analyses. The information obtained through this
structure-function analysis will be critical for establishing sequence and/or structural parameters which may
enable prediction of lncRNA-protein binding activity, thereby advancing our understanding of the biology of
lncRNAs, even those not included in this initial screen.
We will then validate roles for lncRNAs in hematopoiesis and altered TGF activation. Specifically, we will use
CD34+ cells depleted of SBDS and increase (by ectopic expression) or decrease (by knockdown) lncRNAs
and their interacting proteins and test these effects in self renewal, myeloid lineage commitment, proliferation,
cell death, oxidative stress, inflammation and TGF activation. To further test roles for specific lncRNAs in
TGF signaling, we will also test the ability of TGF inhibitors to rescue normal hematopoietic function. We will
identify TGF-responsive genes in affected subpopulations that could be targeted for novel SDS therapies, and
test their function in SDS hematopoiesis. Thus, these studies will (1) identify lncRNAs, proteins, and lncRNA-
protein interactions directing normal and SDS pathogenic myeloid function; (2) develop a platform for
systemically studying lncRNAs; and (3) provide pre-clinical evidence that could support a clinical trial targeting
lncRNA-directed pathways for future SDS therapy.
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