Development of molecular target therapy against chimeric oncogene using siRNA

真核细胞中RNA干扰（RNAi）的发现是近年来分子和细胞生物学研究的重大突破。RNAi机制在基因调控、基因组防御、染色质结构和动力学等方面发挥生物学功能。今天，RNAi对动物新型疾病模型的发展产生了巨大的影响。此外，小干扰RNA （sirna）作为RNA沉默的触发分子，很可能成为治疗各种疾病的宝贵工具。所有已知的人黏液样和圆细胞脂肪肉瘤（MLS/RCLS）都与染色体易位有关。这些染色体易位导致编码嵌合癌蛋白的基因融合，该嵌合癌蛋白由两个相关基因TLS（也称为FUS）或EWS中的一个提供的N端和由CHOP（也称为GADD153）基因提供的C端组成。在本项目中，我利用RNA干扰方法，在体外证明了靶向TLS-CHOP的小干扰RNA （siRNA）耗尽TLS-CHOP蛋白并引起生长抑制。然而，我们在小鼠模型上未检测到生长抑制作用。另一方面，TLS-CHOP是肿瘤特异性转录因子。因此，我正在尝试使用微阵列分析来识别新的治疗靶分子。此外，我还生成了针对TLS/EWS-CHOP癌蛋白独特肽序列的单克隆抗体。在石蜡包埋组织样品中，使用其中一种抗体进行免疫组化分析比巢式RT-PCR检测TLS-CHOP转录本或其基因产物更敏感。因此，我期望该抗体在MLS/RCLS的分子诊断中具有很大的优势。

The discovery of RNA interference (RNAi) in eukaryotic cells is a major breakthrough in the recent progress of the molecular and cellular biology. RNAi machineries exert biological functions in gene regulation, genome defense, chromatin architecture and dynamics. Today, RNAi has had an enormous impact on the development of novel disease models in animals. In addition, small interfering RNAs (siRNAs), the trigger molecules for RNA silencing, are likely to become invaluable tools for the treatment of various diseases.All of known human myxoid and round cell liposarcomas (MLS/RCLS) are associated with chromosomal translocations. These chromosomal translocations lead to gene fusions that encode chimeric oncoproteins consisting of an N terminus contributed by one of two related genes, TLS (also known as FUS) or EWS, and a C terminus contributed by the CHOP (also called GADD153) gene.In this project, using the RNA interference method, I show that TLS-CHOP-targeting small interfering RNAs (siRNA) depleted TLS-CHOP protein and caused growth inhibition in vitro. However, we could not detect growth inhibition effects on mouse models. On the other hand, TLS-CHOP works as a tumor specific transcription factor. Therefore, I am trying to identify the novel therapeutic target molecle using microarray analysis.In addition, I have generated monoclonal antibodies against the unique peptide sequence of TLS/EWS-CHOP oncoproteins. Immunohistochemical analysis using one of the antibodies was more sensitive than the nested RT-PCR for detection of the TLS-CHOP transcripts or its gene products in paraffin-embedded tissue samples. Thus, I expect that the antibody has a great advantage for molecular diagnosis of MLS/RCLS.