RNAi-Based Therapy of Hepatocellular Carcinoma

基于 RNAi 的肝细胞癌治疗

• 批准号: 7105632
• 负责人: KAIYI LI
• 金额: $ 23.14万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7105632
• 关键词: biotechnology; combination cancer therapy; cyclins; gene delivery system; gene expression; gene targeting; gene therapy; hepatocellular carcinoma; immunocytochemistry; immunoprecipitation; laboratory mouse; neoplasm /cancer chemotherapy; neoplasm /cancer therapy; small interfering RNA; terminal nick end labeling; transfection; tumor suppressor genes; western blottings

DESCRIPTION (provided by applicant): The overall goal of this study is to address the therapeutic potential of cyc E siRNA on the treatment of hepatocellular carcinoma (HCC). Our lab recently discovered that cyclin E (cyc E), an oncogene overexpressed in 70% of HCC, played a substantial role in proliferation and cell survival and could serve as a promising therapeutic target for HCC. We also found that overexpressed cyc E could be suppressed up to 90% by siRNA targeting the coding region of cyc E. Depletion of Cyc E in HCC induced significant inhibition of cell growth both in cultured cells and in nude mice. Therefore, we hypothesize that cyc E siRNA may serve as a novel and effective therapeutic agent to treat cyc E-overexpressing HCC. Four specific aims will be carried out to test this hypothesis. (1) To determine the therapeutic effects of cyc E siRNA using both subcutaneous and orthotopic HCC models in mice. For the orthotopic model, HCC cell lines expressing luciferase will be intrahepatically injected into mice to produce tumors in liver. An improved liposomal delivery system (DOTAP:Chol) will be used for siRNA transfer by intratumoral injection or systemic treatment through intravenous injection. The tumor volume and metastasis will be monitored by in vivo image system to determine the therapeutic efficacy. We will also compare the tumor suppression effects among treatments with different delivery systems (non-liver targeting versus liver-targeting delivery system) to develop an optimal preclinical therapeutic strategy for HCC. (2) To examine the effect of cyc E siRNA on HCC cells versus normal hepatocytes or tissues in in vitro and in vivo models. The siRNA will be transfected into immortalized normal human hepatocytes or HCC cells and the growth properties among those cells will be fully analyzed for their differences. The in vivo toxicity of siRNA will be examined by enzymatic and pathological analysis on major organs in mice after the treatments. (3) To evaluate the therapeutic efficacy of cyc E siRNA in combination with chemodrugs. We have demonstrated that combination of cyc E siRNA and doxorubicin exhibited a synergism on inhibition of HCC cell growth. To test if cyc E overexpression is involved in chemoresistance, stable cell lines with different cyc E expression levels will be generated and examined for their responses to multiple chemodrugs. The synergistic effects from the combinations involving cyc E siRNA and chemodrugs will be further tested in animals. We will also identify the mechanisms mediating these synergistic effects, including analysis of NF-kappaB, Akt and Bcl2 survival pathways. (4) To assess the in vitro and in vivo antitumor effect of cyc E siRNA from a tumor specific expression vector. We have generated a plasmid which expresses cyc E siRNA via a liver tumor specific promoter (AFP). The efficacy and specificity of this vector will be systematically assessed using both HCC cell lines and HCC xenograft models in mice. The data generated from this study will provide valuable information to further understanding the molecular events involved in the development of HCC as well as lead to the development of effective cyc E siRNA-based therapy to reduce HCC mortality.

描述（由申请人提供）：本研究的总体目标是探讨 cyc E siRNA 在肝细胞癌 (HCC) 治疗中的治疗潜力。我们的实验室最近发现细胞周期蛋白 E (cyc E) 是一种在 70% 的 HCC 中过度表达的癌基因，在增殖和细胞存活中发挥着重要作用，可以作为 HCC 的有前途的治疗靶点。我们还发现，针对 cyc E 编码区的 siRNA 可以将过表达的 cyc E 抑制高达 90%。 HCC 中 Cyc E 的缺失会导致培养细胞和裸鼠中细胞生长的显着抑制。因此，我们假设 cyc E siRNA 可作为治疗 cyc E 过表达 HCC 的新型有效治疗剂。将实施四个具体目标来检验这一假设。 (1) 使用小鼠皮下和原位 HCC 模型确定 cyc E siRNA 的治疗效果。对于原位模型，表达荧光素酶的 HCC 细胞系将​​被肝内注射到小鼠体内以在肝脏中产生肿瘤。改进的脂质体递送系统（DOTAP：Chol）将用于通过瘤内注射或通过静脉注射进行全身治疗的siRNA转移。通过体内图像系统监测肿瘤体积和转移情况以确定治疗效果。我们还将比较不同递送系统（非肝脏靶向与肝脏靶向递送系统）治疗的肿瘤抑制效果，以制定最佳的 HCC 临床前治疗策略。 (2) 在体外和体内模型中检测cyc E siRNA对HCC细胞与正常肝细胞或组织的影响。 siRNA将被转染到永生化的正常人肝细胞或HCC细胞中，并且将充分分析这些细胞之间的生长特性的差异。 siRNA的体内毒性将通过治疗后小鼠主要器官的酶学和病理学分析来检查。 (3)评价cyc E siRNA联合化疗药物的治疗效果。我们已经证明，cyc E siRNA 和阿霉素的组合在抑制 HCC 细胞生长方面表现出协同作用。为了测试 cyc E 过度表达是否与化疗耐药有关，将生成具有不同 cyc E 表达水平的稳定细胞系，并检查它们对多种化疗药物的反应。 cyc E siRNA 和化疗药物组合的协同效应将在动物身上进行进一步测试。我们还将确定介导这些协同效应的机制，包括分析 NF-kappaB、Akt 和 Bcl2 生存途径。 (4)评估来自肿瘤特异性表达载体的cyc E siRNA的体外和体内抗肿瘤作用。我们生成了一个通过肝肿瘤特异性启动子 (AFP) 表达 cyc E siRNA 的质粒。该载体的功效和特异性将使用 HCC 细胞系和小鼠 HCC 异种移植模型进行系统评估。这项研究产生的数据将为进一步了解 HCC 发展中涉及的分子事件提供有价值的信息，并促进开发有效的基于 cyc E siRNA 的疗法以降低 HCC 死亡率。