Studies of amplification in rhabdomyosarcoma

在过去对融合阳性横纹肌肉瘤(RMS)基因扩增的研究中，我们发现MYCN和MIR17HG分别是2p24和13q31扩增的关键靶点。MYCN编码Myc转录因子家族的一个成员，MIR17HG编码六个microRNA(MiRNA)。在我们最近的研究中，我们研究了PAX3-FOXO1(P3F)与MYCN和MIR17HG的融合关系。为了将这些基因联系在一个共同的途径上，我们注意到，我实验室以前的研究表明，MYCN是P3F上调的转录靶标，而其他已发表的研究表明，Myc家族蛋白，如MYCN，有助于MIR17HG的上调。基于这些发现，我们假设了一种情景，即P3F上调MYCN，进而上调MIR17HG。为了研究这一场景的有效性，我们使用了两种基于细胞培养的系统。在第一个系统中，我们设计了两个FP RMS细胞系(RH30和RH41)，其中含有可诱导的Cas9，并结构性地表达PAX3导向的gRNA，从而允许可诱导的P3F基因敲除。在每个RMS系中，P3F基因敲除导致MYCN表达显著降低。对由MIR17HG处理的miR-17-92簇(miR-17、miR-18、miR-19a、miR-19b、miR-20和miR-92)的6个成熟miRNA的分析也表明，在P3F基因敲除后，这些miRNA的表达显著降低。在救援实验中，慢病毒转导P3F结构(经修改以抵抗基因敲除)到这些P3F基因敲除细胞中，增加了MYCN和miR-17-92簇的miRNA的表达。有趣的是，最近用慢病毒转导MYCN进入P3F基因敲除细胞的实验表明，MYCN可能只恢复了比P3F救援恢复的更低水平的miR-17-92表达。这些发现表明，P3F可能对MYCN未完全概括的MIR17HG(和miR-17-92簇)具有调节作用。在第二个系统中，我们使用人成肌细胞系DBT以及可诱导的P3F和结构性MYCN构建，以评估P3F和/或MYCN对该miRNA簇的影响。应该注意的是，P3F只诱导这些成肌细胞中MYCN的表达小幅增加，因此该系统允许独立评估P3F和MYCN，而不会对彼此的表达产生显著影响。在这些实验中，单独的P3F不能显著上调成肌细胞中miRNA的表达，而MYCN的表达则诱导6个miRNA中的每一个的表达略有增加。相反，P3F和MYCN的结合导致6个miRNA的表达显著增加。因此，P3F和MYCN之间似乎存在协同作用，因此P3F不完全通过上调MYCN起作用。虽然MYCN可能是P3F上调六个microRNA所必需的，但MYCN不足以完全上调这些靶点。为了评估高水平和低水平miR-17-92表达的表型效应，我们开发了两个实验系统。在一个系统中，我们将组成性表达miR-17-92簇的慢病毒构建体导入人DBT成肌细胞系统的衍生物中，导致相应的六个miRNA的表达增加，无论是否增加MYCN或P3F的表达。为了研究功能的丧失，我们设计了人RMS细胞系来表达一个可诱导的Cas9-KRAB-MeCP2结构和一个结构性表达六个针对MIR17HG 5‘调控区的gRNA的结构。在这些细胞中，修饰的Cas9蛋白作为转录抑制因子，下调MIR17HG的表达，导致miR-17-92簇的六个miRNA水平非常低。现在这两个系统已经完全开发和实施，我们将分析与DBT成肌细胞miR-17-92表达增加和人RMS细胞miR-17-92表达降低相关的表型变化。

In past studies of gene amplification in fusion-positive (FP) rhabdomyosarcoma (RMS), we identified MYCN and MIR17HG as key targets of the 2p24 and 13q31 amplicons, respectively. MYCN encodes a member of the Myc family of transcription factors and MIR17HG generates six microRNA's (miRNA's). In our recent studies, we investigated the relationship of the PAX3-FOXO1 (P3F) fusion with MYCN and MIR17HG. To link these genes in a common pathway, we note that previous studies in my laboratory showed that MYCN is a transcriptional target upregulated by P3F, and other published studies revealed that Myc family proteins, such as MYCN, contribute to the upregulation of MIR17HG. Based on these findings, we hypothesized a scenario in which P3F upregulates MYCN, which in turn upregulates MIR17HG. To investigate the validity of this scenario, we used two cell culture-based systems. In the first system, we engineered two FP RMS cell lines (RH30 and RH41) with an inducible Cas9 and constitutively expressing PAX3-directed gRNA's, thereby permitting inducible knockout of P3F. In each RMS line, P3F knockout resulted in a substantial decrease in MYCN expression. Analysis of the six mature miRNA's of the miR-17-92 cluster (miR-17, miR-18, miR-19a, miR-19b, miR-20 and miR-92) processed from MIR17HG also demonstrated a substantial decrease in expression of these miRNA's following P3F knockout. In rescue experiments, lentiviral transduction of a P3F construct (modified to be resistant to knockout) into these P3F knockout cells increased expression of MYCN and the miRNA's of the miR-17-92 cluster. Of interest, recent experiments with lentiviral transduction of MYCN into the P3F knockout cells suggests that MYCN may only restore a lower level of miR-17-92 expression than the level restored by P3F rescue. These findings suggest that P3F may have regulatory effects on MIR17HG (and the miR-17-92 cluster) that are not fully recapitulated by MYCN. In a second system, we used the human myoblast line Dbt along with inducible P3F and constitutive MYCN constructs to assess the effects of P3F and/or MYCN on this miRNA cluster. It should be noted that P3F only induces a small increase in MYCN expression in these myoblasts so that this system permits P3F and MYCN to be independently assessed without significant effects on each other's expression. In these experiments, P3F alone does not significantly upregulate miRNA expression in the myoblasts whereas MYCN expression induces a small increase in expression of each of the six miRNA's. In contrast, the combination of P3F and MYCN results in a substantial increase in expression of each of the six miRNA's. Therefore, there appears to be a synergistic effect between P3F and MYCN so that P3F does not entirely act through upregulation of MYCN. Although MYCN may be necessary for P3F to upregulate the six microRNA's, MYCN is not sufficient to fully upregulate these targets. To assess the phenotypic effects of high versus low level miR-17-92 expression, we developed two experimental systems. In one system, we transduced a lentiviral construct constitutively expressing the miR-17-92 cluster into derivatives of the human Dbt myoblast system, resulting in increased expression of the six corresponding miRNA's with or without increased expression of MYCN or P3F. For loss of function studies, we engineered human RMS cell lines to express an inducible Cas9-KRAB-MeCP2 construct and a construct constitutively expressing six gRNA's targeting the MIR17HG 5' regulatory region. In these cells, the modified Cas9 protein acts as a transcriptional repressor and down-regulates MIR17HG expression, resulting in very low levels of the six miRNA's of the miR-17-92 cluster. Now that these two systems have been fully developed and implemented, we will assay the phenotypic changes associated with increased miR-17-92 expression in Dbt myoblasts and decreased miR-17-92 expression in human RMS cells.