RNAi-Based Therapy of Hepatocellular Carcinoma

描述(由申请人提供)：这项研究的总体目标是探讨Cyc E siRNA在治疗肝细胞癌(HCC)方面的治疗潜力。本实验室最近发现，细胞周期蛋白E(Cyclin E，cyc E)是一种在70%的肝细胞癌中过度表达的癌基因，在细胞增殖和细胞存活中发挥重要作用，有望成为治疗肝细胞癌的靶点。我们还发现，针对Cyc E编码区的siRNA可以抑制Cyc E的过度表达高达90%。Cyc E在肝癌中的缺失导致了对培养细胞和裸鼠细胞生长的显着抑制。因此，我们推测Cyc E siRNA可能成为治疗Cyc E过表达的肝癌的一种新的有效的治疗药物。我们将通过四个具体目标来检验这一假说。(1)采用小鼠皮下和原位肝癌模型，观察Cyc E siRNA对肝癌的治疗作用。对于原位模型，表达荧光素酶的肝癌细胞系将被肝内注射到小鼠体内，以产生肝脏肿瘤。一种改进的脂质体递送系统(DOTAP：CHOL)将用于瘤内注射或静脉注射全身治疗的siRNA转移。通过活体影像系统监测肿瘤体积和转移情况，以确定治疗效果。我们还将比较不同给药系统(非肝靶向给药系统和肝靶向给药系统)的肿瘤抑制效果，以开发出最佳的肝癌临床前治疗策略。(2)通过体内外模型检测Cyc E siRNA对肝癌细胞和正常肝细胞及组织的作用。SiRNA将被导入永生化的正常人肝细胞或肝癌细胞，并将充分分析这些细胞之间的生长特性的差异。治疗结束后，通过对小鼠主要脏器的酶学和病理学分析，检测siRNA的体内毒性。(3)评价Cyc E siRNA联合化疗药物的疗效。我们已经证明了cyc E siRNA和阿霉素联合应用在抑制肝癌细胞生长方面具有协同作用。为了测试Cyc E的过度表达是否与化疗耐药有关，我们将产生具有不同Cyc E表达水平的稳定细胞株，并检测它们对多种化疗药物的反应。涉及cyc E、siRNA和Chemodrugs的组合的协同效应将在动物身上进一步测试。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生存途径的分析。(4)评价肿瘤特异性表达载体cyc E siRNA的体内外抗肿瘤作用。我们已经制备了一个通过肝癌特异性启动子(AFP)表达Cyc E siRNA的质粒。该载体的有效性和特异性将使用肝癌细胞系和小鼠肝癌异种移植模型进行系统评估。这项研究的数据将为进一步了解肝癌发生发展中涉及的分子事件提供有价值的信息，并有助于开发有效的基于cyc E siRNA的治疗方法来降低肝癌死亡率。

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.