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

描述（由申请人提供）：横纹肌肉瘤（Rhabdomyosarcoma， 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种肿瘤类型中过表达，其中包括对转移至关重要的RMS。Six1的表达通常局限于胚胎早期发育，其促进前体细胞的激活和迁移有助于肌肉、肾脏和内耳的正常形成。在斑马鱼中有两个SIX1同源物，six1a和six1b，对six1a的作用知之甚少。由于发育过程会促进肿瘤的发生，因此对胚胎发生过程中six1功能的研究引起了人们极大的兴趣。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肉发生过程中，microRNAs （miRs）已被证明能够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基因的调控。在肿瘤模型中，mir被证明可以抑制RMS肿瘤的生长，并且mir介导的Six1下调可以阻止肾肿瘤的进展。这些数据表明，在肌生成中研究靶向six1的miRs可以为RMS的新疗法提供见解。目的：利用斑马鱼评估six1a/b的功能，并研究mir介导的six1在肌肉发生和肿瘤发生过程中的调节。假设：six1a和six1b是肌发生所必需的，它们的表达受到miRs 30a、216a和219-3p的严格控制。six1的异常表达可能通过miR下调促进斑马鱼RMS发病。这些miRs的异位表达将导致six1下调并抑制RMS的进展。具体目的：1)研究six1a/b在斑马鱼肌肉发育过程中的功能。2)确定miRs 30a、219-3p和/或216a是否在斑马鱼肌发生过程中调控six1a/b。3)探讨six1a/b和miRs 30a、216a和/或219-3p在RMS发生和进展中的作用。通过注射1-4个细胞胚胎，利用morpholino介导的敲低和mRNA/miR过表达来操纵斑马鱼体内基因表达，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等形态学分析来评估表型。我们将在建立的斑马鱼RMS模型中分析six1a/b和调节性miRs的潜在作用，并通过在rag2阳性肌肉祖细胞中驱动six1a/b过表达的模型开发进一步探索。我们将分析SIX1和miR在人RMS肿瘤中的表达，以评估疾病分期的相关性。许多发育基因促进细胞特性，如生存和迁移。这些基因在发育过程中被下调，但在癌细胞中经常被重新激活。研究six1a和six1b在胚胎发生和肿瘤发生过程中是如何被控制的，不仅可以为RMS患者提供治疗干预，也可以为那些患有SIX1活跃的众多癌症的患者提供治疗干预。

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.