Role of microR-122 in Hepatocarcinogenesis using Conditional Knockout Mice

描述（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，如肝炎、纤维化和肝硬化的并发症而发生。鉴定直接或间接促成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。其中一个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核苷酸）非编码调节RNA的下调与预后不良、肿瘤复发和转移有关。我们发现，在小鼠中，破坏miR-122在肝脏中的作用会导致高渗透性HCC的发展。这些肝脏特异性miR-122敲除（LKO）小鼠自发发展为肝炎、纤维化和肝细胞癌，并伴有肺转移。更重要的是，我们和其他人发现，通过病毒载体（AAV8）或脂质体纳米颗粒递送miR-122可以抑制不同小鼠模型中的HCC发展，这表明miR-122在HCC患者中具有治疗潜力。我们现在建议使用我们的新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。Aim 1将(a)使用miR-122KO肝脏作为阴性对照，通过无偏倚的hit - clip和RNA-seq方法验证在野生型肝脏转录组中鉴定出的miR-122的功能靶点，以及(b)研究miR-122与原发性人类HCC中这些靶点之间的反相关性。我们将重点关注在不同阶段促进肿瘤发生的靶点。目的2将测试抗炎（Ccl2）的治疗效果

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.