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

• 批准号: 8455796
• 负责人: Jenean O'Brien
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 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/8455796
• 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

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（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%。促转移同源异型盒（Hox）转录因子SIX 1在至少10种肿瘤类型中过表达，包括RMS，其中它对转移至关重要。Six 1的表达通常限于早期胚胎发育，其促进前体细胞活化和迁移有助于肌肉、肾脏和内耳的正常形成。在斑马鱼中有两个SIX 1同源物，six 1a和six 1b，对six 1a的作用知之甚少。由于发育程序促进肿瘤发生，因此研究six 1在胚胎发生过程中的功能具有重要意义。此外，在发育和肿瘤发生过程中控制six 1表达的机制还有待阐明。在整个胚胎发生和肌肉发生过程中，microRNAs（miRs）已被证明可以协调复杂的时间和组织特异性蛋白表达模式，包括许多Hox基因的调控。在肿瘤模型中，miR显示出抑制RMS肿瘤生长，并且miR介导的Six 1下调可以防止肾脏肿瘤进展。这些数据表明，在肌发生中靶向six 1的miR的研究可以为RMS的新疗法提供见解。目的：利用斑马鱼研究six 1 a/B的功能，并探讨miR介导的six 1在肌肉发生和肿瘤发生中的调控作用。假设：six 1a和six 1b是肌肉发生所必需的，它们的表达受到miRs 30 a，216 a和219- 3 p的严格控制。six 1的异常表达，可能通过miR下调，可以促进斑马鱼RMS的发病。这些miR的异位表达将导致six 1下调和RMS进展的抑制。具体目的：1）研究斑马鱼肌肉发育过程中6个1a/B基因的功能。2)确定miR 30 a、219- 3 p和/或216 a是否在斑马鱼肌发生过程中调节six 1 a/B。3)研究six 1 a/B和miR 30 a、216 a和/或219- 3 p在RMS发生和进展中的作用。通过注射到1-4细胞胚胎中的吗啉代介导的敲低和mRNA/miR过表达将用于操纵斑马鱼中的体内基因表达，随后进行原位杂交、实时PCR mRNA定量、免疫印迹和免疫荧光蛋白检测以及其他形态学分析以评估表型。six 1a/B和调节miR的潜在作用将在建立的斑马鱼RMS模型中进行分析，并通过驱动rag 2阳性肌肉祖细胞中six 1a/B过表达的模型开发进行进一步探索。将分析人RMS肿瘤中的SIX 1和miR表达以评估疾病分期相关性。许多发育基因促进细胞特性，如存活和迁移。这些基因在发育过程中下调，但通常在癌细胞中重新激活。研究six 1a和six 1b在胚胎发生和肿瘤发生过程中是如何被控制的，可以为治疗干预提供洞察力，不仅适用于RMS患者，而且适用于那些患有SIX 1活跃的众多癌症中的任何一种的患者。