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（由申请方提供）：本研究的总体目标是探讨cyc E siRNA治疗肝细胞癌（HCC）的治疗潜力。我们的实验室最近发现，细胞周期蛋白E（cyc E），在70%的HCC中过表达的癌基因，在增殖和细胞存活中发挥了重要作用，并可能作为HCC的一个有前途的治疗靶点。我们还发现，通过siRNA靶向cyc E的编码区，过表达的cyc E可以被抑制高达90%。肝癌细胞中Cyc E的耗尽在培养细胞和裸鼠中均诱导细胞生长的显著抑制。因此，我们推测，cyc E siRNA可能作为一种新的和有效的治疗剂，以治疗cyc E过表达的肝癌。将进行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，将表达荧光素酶的HCC细胞系肝内注射到小鼠中以在肝脏中产生肿瘤。将使用改进的脂质体递送系统（DOTAP：Chol）通过瘤内注射或通过静脉内注射的全身治疗进行siRNA转移。通过体内成像系统监测肿瘤体积和转移，以确定治疗效果。我们还将比较不同给药系统（非肝靶向与肝靶向给药系统）治疗的肿瘤抑制效果，以制定HCC的最佳临床前治疗策略。(2)在体外和体内模型中检测cyc E siRNA对HCC细胞相对于正常肝细胞或组织的作用。将siRNA转染到永生化的正常人肝细胞或HCC细胞中，并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对小鼠主要器官的酶和病理学分析来检查。(3)目的评价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过表达是否参与化学抗性，将产生具有不同cyc E表达水平的稳定细胞系，并检查其对多种化学药物的应答。将在动物中进一步测试来自涉及cyc E siRNA和化学药物的组合的协同效应。我们还将确定介导这些协同效应的机制，包括分析NF-κ B，Akt和Bcl-2生存途径。(4)目的：研究肿瘤特异性表达载体cyc E siRNA的体内外抗肿瘤作用。我们已经产生了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细胞系和小鼠中的HCC异种移植物模型进行系统评估。这项研究产生的数据将提供有价值的信息，以进一步了解参与HCC发展的分子事件，并导致开发有效的基于cyc E siRNA的治疗方法，以降低HCC死亡率。