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）：这项研究的总体目标是解决Cyc e siRNA在治疗肝细胞癌（HCC）方面的治疗潜力。我们的实验室最近发现，在70％的HCC中过表达的癌基因Cyclin E（Cyc E）在增殖和细胞存活中发挥了重要作用，可以作为HCC的有希望的治疗靶标。我们还发现，通过靶向CYC E的siRNA，可以抑制过表达的CYC E。在HCC中，CYC E的编码区域的siRNA诱导了培养细胞和裸小鼠的细胞生长的显着抑制。因此，我们假设Cyc e siRNA可以用作一种新型且有效的治疗剂，以治疗CYC e Over表达HCC。将进行四个具体目标以检验这一假设。 （1）使用小鼠的皮下和原位HCC模型来确定CYC E siRNA的治疗作用。对于原位模型，表达荧光素酶的HCC细胞系将在肝脏内注射到小鼠中，以在肝脏中产生肿瘤。改进的脂质体递送系统（DOTAP：CHOL）将通过肿瘤内注射或全身治疗通过静脉注射来转移siRNA。肿瘤体积和转移将通过体内图像系统监测，以确定治疗功效。我们还将比较不同的递送系统（非肝脏靶向与肝脏靶向输送系统）的处理之间的肿瘤抑制作用，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查Cyc e siRNA对HCC细胞与正常肝细胞或组织的影响。 siRNA将被转染到永生的正常人肝细胞或HCC细胞中，这些细胞之间的生长特性将被充分分析其差异。 siRNA的体内毒性将通过对治疗后小鼠的主要器官进行酶促和病理分析来检查。 （3）评估Cyc e siRNA与化学戒指结合使用的治疗功效。我们已经证明，Cyc e siRNA和阿霉素的组合表现出有关抑制HCC细胞生长的协同作用。为了测试CYC E是否参与化学抗性，将生成具有不同CYC E表达水平的稳定细胞系，并检查其对多种化学果的反应。涉及Cyc e siRNA和化学果的组合产生的协同作用将在动物中进一步测试。我们还将确定介导这些协同作用的机制，包括对NF-kappab，AKT和BCl2存活途径的分析。 （4）评估Cyc e siRNA的体外和体内抗肿瘤效应。我们已经产生了一种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在小鼠中使用HCC细胞系和HCC异种移植模型系统地评估。这项研究产生的数据将提供有价值的信息，以进一步了解HCC发展所涉及的分子事件，并导致开发有效的基于CYC E SIRNA的治疗以降低HCC死亡率。