Regulation and function of Six1 in zebrafish muscle development and in rhabdomyos

Six1 在斑马鱼肌肉发育和横纹肌中的调节和功能

基本信息

批准号：
8774835
负责人：
Jenean O'Brien
金额：
$ 5.51万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-02-01 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8774835
关键词：
Accounting Alveolar Animal Model Automobile Driving Biological Assay Birth Brain Breast Cell physiology Cells Cervical Child Childhood Colon Complex Data Detection Development Developmental Gene Diagnosis Disease Disease Progression Down-Regulation Ectopic Expression Embryo Embryonic Development Fishes Gene Expression Genes Homeobox Homeobox Genes Homologous Gene Human Immunoblotting Immunofluorescence Immunologic In Situ Hybridization Injection of therapeutic agent Investigation Kidney Kidney Neoplasms Labyrinth Lead Lung Malignant Childhood Neoplasm Malignant Neoplasms Mediating Messenger RNA MicroRNAs Modeling Mus Muscle Muscle Development Muscle satellite cell Myoblasts Neoplasm Metastasis Orthologous Gene Ovarian Patients Pattern Pectoral Phenotype Population Property Proteins Regulation Reporter Repression Research Rhabdomyosarcoma Role Sampling Staging Stem cells Survival Rate Therapeutic Intervention Time Tissues Transgenic Organisms Tumor Promotion Zebrafish cancer cell cancer initiation design driving force embryo cell gene function genetic manipulation in vivo insight interest malignant muscle neoplasm migration model development mouse model myogenesis neoplastic cell novel overexpression postnatal precursor cell prevent progenitor programs protein expression public health relevance research study sarcoma satellite cell soft tissue therapeutic target therapy design therapy development transcription factor tumor tumor growth tumor progression tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Rhabdomyosarcoma (RMS) accounts for ~8% of all pediatric cancers, where the overall 5-year survival rate for children diagnosed with metastatic RMS is less than 30%. The pro-metastatic Homeobox (Hox) transcription factor SIX1 is overexpressed in at least 10 tumor types, including RMS, where it is critical for metastasis. Six1 expression is normally limited to early embryonic development, where its promotion of precursor cell activation and migration contribute to proper formation of muscle, kidney and the inner ear. In zebrafish there are two SIX1 homologs, six1a and six1b, with little known about the role of six1a. Because developmental programs promote tumorigenesis, investigation of six1 function during embryogenesis is of great interest. Further, the mechanisms controlling six1 expression during development and tumorigenesis are yet to be elucidated. Throughout embryogenesis and myogenesis, microRNAs (miRs) have been shown to coordinate complex temporal and tissue-specific patterns of protein expression, including regulation of many Hox genes. In tumor models, miRs are shown to inhibit RMS tumor growth, and miR-mediated downregulation of Six1 can prevent kidney tumor progression. These data indicate that investigation of miRs targeting six1 in myogenesis could provide insight into new therapies for RMS. Objectives: To utilize zebrafish to assess six1a/b function and to investigate miR-mediated six1 regulation during myogenesis and tumorigenesis. Hypothesis: six1a and six1b are necessary for myogenesis, where their expression is tightly controlled by miRs 30a, 216a, and 219-3p. Aberrant expression of six1, perhaps through miR downregulation, can promote RMS onset in zebrafish. Ectopic expression of these miRs will lead to six1 downregulation and inhibition of RMS progression. Specific aims: 1) To investigate the six1a/b functions during zebrafish muscle development. 2) To determine whether miRs 30a, 219-3p and/or 216a regulate six1a/b during zebrafish myogenesis. 3) To investigate the roles of six1a/b and miRs 30a, 216a and/or 219-3p in RMS initiation and progression. Morpholino-mediated knockdown and mRNA/miR overexpression through injection into 1-4 cell embryos will be utilized to manipulate in vivo gene expression in zebrafish, followed by in situ hybridization, real-time PCR mRNA quantification, immunoblot and immunofluorescent protein detection, and other morphologic analyses to assess phenotypes. Potential roles for six1a/b and regulatory miRs will be analyzed in an established zebrafish RMS model, and further explored through model development by driving six1a/b overexpression in rag2-positive muscle progenitors. SIX1 and miR expression in human RMS tumors will be analyzed to assess disease stage correlation. Many developmental genes promote cell properties, such as survival and migration. These genes are downregulated during development, but are often re-activated in cancer cells. Research into how six1a and six1b are controlled during both embryogenesis and tumorigenesis can provide insight into therapeutic interventions, not only for RMS patients, but for those afflicted with any of the numerous cancers where SIX1 is active.

描述（由申请人提供）：横纹肌肉瘤 (RMS) 约占所有儿科癌症的 8%，其中诊断为转移性 RMS 的儿童的总体 5 年生存率低于 30%。促转移同源盒 (Hox) 转录因子 SIX1 在至少 10 种肿瘤类型中过度表达，其中包括对转移至关重要的 RMS。 Six1 表达通常仅限于早期胚胎发育，其促进前体细胞活化和迁移有助于肌肉、肾脏和内耳的正确形成。斑马鱼中有两个 SIX1 同源物， Six1a 和 Six1b，但人们对 Six1a 的作用知之甚少。由于发育程序会促进肿瘤发生，因此对胚胎发生过程中的 Six1 功能的研究引起了极大的兴趣。此外，在发育和肿瘤发生过程中控制 Six1 表达的机制尚未阐明。在整个胚胎发生和肌发生过程中，microRNA (miR) 已被证明可以协调复杂的时间和组织特异性蛋白质表达模式，包括许多 Hox 基因的调节。在肿瘤模型中，miR 被证明可以抑制 RMS 肿瘤生长，并且 miR 介导的 Six1 下调可以预防肾肿瘤进展。这些数据表明，对肌生成中靶向 61 的 miR 进行研究可以为 RMS 的新疗法提供见解。目的：利用斑马鱼评估 Six1a/b 功能并研究 miR 介导的 Six1 在肌生成和肿瘤发生过程中的调控。假设：six1a 和 Six1b 对于肌生成是必需的，它们的表达受到 miR 30a、216a 和 219-3p 的严格控制。 Six1 的异常表达（可能是通过 miR 下调）可以促进斑马鱼 RMS 的发生。这些 miR 的异位表达将导致 RMS 进展的下调和抑制。具体目标：1）研究斑马鱼肌肉发育过程中的six1a/b功能。 2)确定miRs 30a、219-3p和/或216a是否在斑马鱼肌生成过程中调节six1a/b。 3) 研究 Six1a/b 和 miR 30a、216a 和/或 219-3p 在 RMS 起始和进展中的作用。通过注射到 1-4 个细胞胚胎中吗啉介导的敲低和 mRNA/miR 过表达将用于操纵斑马鱼的体内基因表达，然后进行原位杂交、实时 PCR mRNA 定量、免疫印迹和免疫荧光蛋白检测等形态学分析以评估表型。将在已建立的斑马鱼 RMS 模型中分析 Six1a/b 和调节 miR 的潜在作用，并通过模型开发通过驱动 rag2 阳性肌肉祖细胞中的 Six1a/b 过度表达来进一步探索。将分析人类 RMS 肿瘤中的 SIX1 和 miR 表达，以评估疾病阶段的相关性。许多发育基因促进细胞特性，例如存活和迁移。这些基因在发育过程中被下调，但通常在癌细胞中重新激活。研究 Six1a 和 Six1b 在胚胎发生和肿瘤发生过程中如何受到控制，可以深入了解治疗干预措施，不仅适用于 RMS 患者，也适用于那些患有 SIX1 活跃的众多癌症中的任何一种的患者。