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中发挥生物学功能。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（small interfering RNA，siRNA）作为RNA沉默的触发分子，有可能成为治疗多种疾病的重要工具。所有已知的人类粘液样和圆细胞脂肪肉瘤（myxoid and round cell liposarcomas，MLS/RCLS）都与染色体易位有关。这些染色体易位导致基因融合，编码由两个相关基因之一TLS贡献的N末端组成的嵌合癌蛋白（也称为FUS）或EWS，以及由CHOP贡献的C末端。（也称为GADD 153）基因。本项目利用RNA干扰方法，我表明，TLS-CHOP靶向小干扰RNA（siRNA）耗尽TLS-CHOP蛋白，并在体外引起生长抑制。然而，我们无法检测到对小鼠模型的生长抑制作用。另一方面，TLS-CHOP作为肿瘤特异性转录因子起作用。因此，我尝试利用基因芯片技术来鉴定新的治疗靶分子，并制备了针对TLS/EWS-CHOP癌蛋白独特肽段序列的单克隆抗体。使用其中一种抗体的免疫组织化学分析比巢式RT-PCR更敏感，用于检测石蜡包埋组织样本中的TLS-CHOP转录本或其基因产物。因此，本研究所制备的抗体在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.