Role of microRNA-122 in hepatocarcinogenesis using conditional knock out mice

描述(申请人提供)：这个新的R01申请的目的是促进我们对miR-122在肝癌发生中的作用的理解。肝癌是第五种最常见的癌症，也是癌症相关死亡的第三种常见原因。在美国，肝癌的发病率正在上升，通过化疗、放射或其他传统癌症疗法治愈或治疗的希望微乎其微。其主要风险因素是感染B型和C型肝炎病毒，以及接触包括酒精在内的有毒化学物质，所有这些都会导致慢性肝脏损伤和炎症。利用肝癌发生的动物模型，我们首次证明了miR-122在肝细胞癌的发生和发展过程中以及在人类原发肝癌中的下调。miR-122是肝脏中含量最丰富的肝脏特异性microRNA(约占总miRNA的70%)。MiR-122的抑制是肝细胞癌预后不良和转移的标志。对培养的肝癌细胞的研究表明，miR-122在体外和裸鼠体内都具有肿瘤抑制作用。为了了解miR-122的生物学功能，特别是在肝癌发生过程中，我们产生了条件基因敲除小鼠(由PI获得R21拨款支持)。当这些小鼠与AlbCre小鼠杂交时，miR-122的表达减少了100倍，并随着年龄的增长自发地在肝脏中患上肝炎，这是在喂养了促进肝癌发生的胆碱缺乏的饮食后促成的。更重要的是，miR-122缺失(KO)小鼠在暴露于二乙基亚硝胺时更容易感染肝癌，二乙基亚硝胺是一种有效的致癌物质。基于这些观察，我们假设miR-122在维持肝功能方面起着关键作用，而miR-122的缺失容易导致包括癌症在内的肝脏疾病。该提案的具体目的是：目的1.研究miR-122在喂养胆碱缺乏饮食所致的非酒精性脂肪性肝病(NAFLD)相关肝癌小鼠模型中的作用。1a)将通过比较miR-122/(KO)和miR-122fl/fl(对照)小鼠之间的肝损伤(细胞凋亡、脂肪变性或脂肪肝、炎症、纤维化)和肝肿瘤(腺瘤和癌的形成)来检查它们对CDAA饮食的敏感性，以及1b)将评估miR-122靶标的参与程度。目的2.探讨miR-122在二乙基亚硝胺(DEN)诱导的小鼠肝癌发生中的作用。将监测注射DEN的小鼠的病理/分子变化，如目标1所述。目的3.在DEN模型中，检测miR-122单独或联合化疗药物抑制体内肿瘤生长的治疗潜力。在肿瘤发展的早期阶段(通过核磁共振成像)，小鼠将每周注射一次装载有半乳糖化纳米颗粒(以特异性靶向肝癌细胞)的miR-122模拟物，并将肿瘤生长的消退与混乱的RNA纳米颗粒治疗的小鼠进行比较。这项研究将阐明最丰富的肝脏特异性microRNA在维持正常肝脏生理方面的功能，以及它在动物模型中对肝细胞癌的治疗效果。

DESCRIPTION (provided by applicant): The objective of this new R01 application is to advance our understanding of the role of miR-122 in hepatocarcinogenesis. HCC is the fifth most common cancer and the third common cause of cancer related death. The incidence of HCC is on the rise in USA, with little hope for cure or treatment through chemotherapy, radiation or other traditional cancer treatments. Its major risk factors are infection with hepatitis B and C viruses, and exposure to toxic chemicals, including alcohol, all of which cause chronic liver injury and inflammation. Using an animal model for hepatocarcinogenesis we were the first to demonstrate down regulation of miR-122, the most abundant liver-specific microRNA (~70% of the total miRNA) in the liver, during the initiation and progression of HCC and also in human primary HCCs. Suppression of miR-122 is a signature of HCCs with poor prognosis and metastasis. Studies with HCC cells in culture have shown that miR-122 functions as a tumor suppressor in vitro and in nude mice. To understand the biological functions of miR-122, especially in hepatocarcinogenesis, we have generated conditional knockout mice (supported by an R21 grant to the PI). These mice express 100 fold less miR-122 when crossed to AlbCre mice and spontaneously develop hepatitis in the liver with age, which is facilitated after feeding choline-deficient diet that promotes hepatocarcinogenesis. More importantly, miR-122 deleted (KO) mice are more susceptible to HCCs when exposed to diethylnitrosamine, a potent liver carcinogen. Based on these observations we hypothesize that miR-122 plays a critical role in maintaining liver function, and loss of miR-122 predisposes to liver disease including cancer. The specific aims of the proposal are: Aim 1. Investigate the role of miR-122 in a mouse model of nonalcoholic fatty liver disease (NAFLD) related HCC induced by feeding choline-deficient diet. 1a) The susceptibility of miR-122 / (KO) and miR-122fl/fl (control) mice to CDAA diet will be examined by comparing liver damage (apoptosis, steatosis or fatty liver, inflammation, fibrosis) and liver tumors (formation of adenomas and carcinomas) between these mice, and 1b) the involvement of miR-122 targets will be assessed. Aim 2. Investigate the role of miR-122 in diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Pathological/molecular changes of mice injected with DEN will be monitored as described in Aim 1. Aim 3. Examine the therapeutic potential of miR-122 alone or in combination with chemotherapeutic agents to inhibit tumor growth in vivo in the DEN model. Mice will be injected weekly for 4 weeks with miR-122 mimetics loaded in galactosylated nanoparticles (to specifically target it to HCC cells) at early stages of tumor development (visualized by MRI) and the regression in the tumor growth will be compared to those in mice treated with the scrambled RNA nanoparticles. This study will elucidate the function of the most abundant liver-specific microRNA in maintaining normal liver physiology and also its therapeutic efficacy against hepatocellular carcinomas in an animal model.