Role of microR-122 in Hepatocarcinogenesis using Conditional Knockout Mice

microR-122 在条件性基因敲除小鼠肝癌发生中的作用

• 批准号: 9197407
• 负责人: Kalpana Ghoshal
• 金额: $ 6.15万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-17 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9197407
• 关键词: Affect; Alcohols; Anti-Inflammatory Agents; Anti-inflammatory; Antineoplastic Agents; Apoptosis; Attenuated; Bioavailable; Biological Process; Cancer Immunology Science; Cells; Cirrhosis; Clinical Trials; Complication; Data; Development; Down-Regulation; Drug Targeting; Drug usage; Energy-Generating Resources; Enzymes; Fibrosis; Future; Gene Expression; Germ Lines; Glutamates; Glutaminase; Glutamine; Goals; Growth; Health; Hepatic; Hepatitis; Hepatitis B Virus; Hepatitis C virus; Hepatocarcinogenesis; Hepatocyte; Homeostasis; Human; Incidence; Inflammation; Inflammatory; Iron; Knock-out; Knockout Mice; Liposomes; Liver; Liver Fibrosis; Liver diseases; Liver neoplasms; Maintenance; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of liver; Metabolic Pathway; Metabolism; Metastatic Neoplasm to the Lung; MicroRNAs; Modeling; Molecular; Mus; Neoplasm Metastasis; Nucleotides; Oligonucleotides; Pathogenesis; Pathway interactions; Patients; Penetrance; Pharmaceutical Preparations; Phase I Clinical Trials; Play; Primary carcinoma of the liver cells; Proto-Oncogenes; RNA; Recurrence; Regulation; Role; Source; Staging; Testing; Therapeutic; Tissues; Treatment Efficacy; Treatment Protocols; Tumor Burden; Tumor Suppressor Proteins; United States; Untranslated RNA; Up-Regulation; Viral Vector; Work; Xenograft Model; attenuation; base; c-myc Genes; cancer cell; chemotherapeutic agent; clinically relevant; crosslinking and immunoprecipitation sequencing; derepression; design; drug development; effective therapy; hepatocellular carcinoma cell line; improved; ineffective therapies; inhibitor/antagonist; insight; liver function; meetings; metabolomics; mortality; mouse model; nanoparticle; neutralizing antibody; nonalcoholic steatohepatitis; novel; novel therapeutics; outcome forecast; pre-clinical; research clinical testing; response; small molecule; transcriptome; transcriptome sequencing; tumor; tumorigenesis; tumorigenic

DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC), the most prevalent liver cancer, ranks third in cancer-related mortality because of ineffective therapy. HCC almost always arises due to complication of underlying liver disease, e.g. hepatitis, fibrosis and cirrhosis. Identification of cellular and molecular entities that directly or indirectly contributeto HCC development will be central to achieve the goal of developing effective therapy. One such molecule that is critical to the maintenance of normal liver function is miR-122, the most abundant liver-specific microRNA. Down regulation of this small (22-nucleotide) non-coding regulatory RNA is associated with poor prognosis, tumor recurrence and metastasis. We found that in mice, disruption of miR-122 action in liver results in development of highly penetrant HCC. These liver-specific miR-122 knockout (LKO) mice spontaneously develop hepatitis, fibrosis and HCC with lung metastasis. More importantly, others and we found that miR-122 delivery via the viral vector (AAV8) or liposomal nanoparticles inhibited HCC development in different mouse models, suggesting a therapeutic potential of miR-122 in HCC patients. We now propose to use our novel mouse model to delineate the underlying mechanism of hepatitis, fibrosis and hepatocarcinogenesis, and to develop effective therapeutics for liver cancer. To this end, we propose the following aims. Aim 1 will (a) validate functional targets of miR-122 that are identified in the wild type liver transcriptome by unbiased HITS-CLIP and RNA-seq approach using miR-122KO livers as a negative control, and (b) investigate anti-correlation between miR-122 and these targets in primary human HCC. We will focus on the targets that promote tumorigenesis at different stages. Aim 2 will test therapeutic efficacy of anti- inflammatory (Ccl2 neutralizing Ab) and anti-fibrotic (Ctgf neutralizing Ab) agents in combination with miR-122 delivered using HCC-targeted liposomal nanoparticles. We will focus on the Ccl2-Ccr2 axis since our supporting data showed that blocking this axis reduced inflammation, fibrosis and tumor burden in these mice, and on Ctgf because it is a direct target of miR-122 identified by HITS-CLIP. Studies proposed here will elucidate the mechanism of suppression of hepatic fibrosis and hepatocarcinogenesis by blocking functions of Ccl2 and Ctgf in combination with miR-122. Aim 3 will decipher the role of the cellular oncogene c-Myc and its target glutaminase 1 (Gls1) that deamidates glutamine to glutamate, a key energy source to cancer cells, in HCC development in LKO mice using clinically relevant c-Myc and Gls1 inhibitors. This aim draws on our finding that blocking c-Myc gene expression, using the small molecule drug JQ1, inhibited growth, clonogenic survival and promoted apoptosis of cultured human HCC cells. Further, using isotopologue studies, we found that LKO livers and tumors show increased conversion of glutamine to glutamate that correlated with an increase in c-Myc-regulated Gls1, an enzyme facilitating utilization of glutamine as an energy source. Collectively, the proposed studies will generate pre-clinical results that will be key for designing future clinical trials with one or mor of the above chemotherapeutic agents in combination with miR-122 for treatment of patients with HCC.

描述（由申请人提供）：肝细胞癌（HCC）是最常见的肝癌，由于治疗无效，在癌症相关死亡率中排名第三。HCC几乎总是由于潜在的肝脏疾病的并发症而发生，例如肝炎、纤维化和肝硬化。识别直接或间接促进HCC发展的细胞和分子实体将是实现开发有效治疗目标的核心。其中一种对维持正常肝功能至关重要的分子是miR-122，这是最丰富的肝脏特异性microRNA。这种小的（22个核苷酸）非编码调节RNA的下调与不良预后、肿瘤复发和转移相关。我们发现，在小鼠中，肝脏中miR-122作用的破坏导致高度渗透性HCC的发展。这些肝脏特异性miR-122敲除（LKO）小鼠自发地发展肝炎、纤维化和具有肺转移的HCC。更重要的是，其他人和我们发现，通过病毒载体（AAV8）或脂质体纳米颗粒递送miR-122抑制了不同小鼠模型中HCC的发展，表明miR-122在HCC患者中的治疗潜力。我们现在建议使用我们的新的小鼠模型来描述肝炎，纤维化和肝癌发生的潜在机制，并开发有效的肝癌治疗方法。为此，我们提出以下目标。目的1将（a）使用miR-122 KO肝脏作为阴性对照，通过无偏HITS-CLIP和RNA-seq方法验证在野生型肝脏转录组中鉴定的miR-122的功能靶标，以及（B）研究原发性人HCC中miR-122与这些靶标之间的反相关性。我们将重点关注在不同阶段促进肿瘤发生的靶点。目的2：检测CCl2的抗炎作用 在一些实施方案中，使用靶向HCC的脂质体纳米颗粒递送与miR-122组合的抗纤维化剂（Ctgf中和Ab）和抗纤维化剂（Ctgf中和Ab）。我们将关注Ccl2-Ccr2轴，因为我们的支持数据表明阻断该轴可减少这些小鼠的炎症，纤维化和肿瘤负荷，并关注Ctgf，因为它是HITS-CLIP鉴定的miR-122的直接靶点。本文提出的研究将阐明通过阻断Ccl2和Ctgf的功能与miR-122联合抑制肝纤维化和肝癌发生的机制。目的3将使用临床相关的c-Myc和Gls1抑制剂，破译细胞癌基因c-Myc及其靶点谷氨酰胺酶1（Gls1）在LKO小鼠HCC发展中的作用，Gls1将谷氨酰胺脱酰胺转化为谷氨酸，这是癌细胞的关键能量来源。这一目的是基于我们的发现，即使用小分子药物JQ1阻断c-Myc基因表达，抑制了培养的人HCC细胞的生长、克隆形成存活并促进了细胞凋亡。此外，使用同位素研究，我们发现LKO肝脏和肿瘤显示谷氨酰胺向谷氨酸的转化增加，这与c-Myc调节的Gls1的增加相关，Gls1是一种促进谷氨酰胺作为能量来源的酶。总的来说，所提出的研究将产生临床前结果，这将是设计未来临床试验的关键，该临床试验使用一种或多种上述化疗剂与miR-122组合治疗HCC患者。