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

描述（由申请人提供）：这项新的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。HCC是癌症相关死亡的第五大常见癌症和第三大常见原因。HCC的发病率在美国呈上升趋势，通过化疗、放疗或其他传统癌症治疗方法治愈或治疗的希望渺茫。其主要危险因素是感染乙型和丙型肝炎病毒，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损伤和炎症。通过肝癌发生的动物模型，我们首次证明了miR-122的下调，miR-122是肝脏中最丰富的肝脏特异性microRNA（约占总microRNA的70%），在HCC的发生和发展过程中以及在人类原发性HCC中也是如此。miR-122的抑制是hcc预后不良和转移的标志。对培养的HCC细胞的研究表明，miR-122在体外和裸鼠中具有肿瘤抑制作用。为了了解miR-122的生物学功能，特别是在肝癌发生中的功能，我们产生了条件敲除小鼠（由PI的R21资助）。当这些小鼠与AlbCre小鼠交叉时，miR-122的表达减少了100倍，随着年龄的增长，肝脏自发发生肝炎，这是在喂食促进肝癌发生的胆碱缺乏饮食后促进的。更重要的是，miR-122缺失（KO）小鼠在暴露于二乙基亚硝胺（一种强致癌物）时更容易发生hcc。基于这些观察结果，我们假设miR-122在维持肝功能中起关键作用，miR-122的缺失易导致包括癌症在内的肝脏疾病。该建议的具体目标是：目标1。研究miR-122在饲喂胆碱缺乏饮食诱导的非酒精性脂肪性肝病（NAFLD）相关HCC小鼠模型中的作用。1a)将通过比较这些小鼠之间的肝损伤（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miR-122 / （KO)和miR-122fl/fl（对照）小鼠对CDAA饮食的易感性，1b）将评估miR-122靶点的参与情况。目标2。探讨miR-122在二乙基亚硝胺（DEN）诱导的肝癌发生中的作用。如目的1所述，观察小鼠注射DEN后的病理/分子变化。目标3。在DEN模型中检测miR-122单独或与化疗药物联合抑制肿瘤生长的治疗潜力。在肿瘤发展的早期阶段（通过MRI观察），每周向小鼠注射含有半乳糖化纳米颗粒的miR-122模拟物（专门针对HCC细胞），并将肿瘤生长的消退与用混乱的RNA纳米颗粒治疗的小鼠进行比较，持续4周。本研究将在动物模型中阐明最丰富的肝脏特异性microRNA在维持肝脏正常生理方面的功能及其对肝细胞癌的治疗作用。

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. PUBLIC HEALTH RELEVANCE: Layman's Abstract The urgent need for new treatments for hepatocellular cancer (HCC) is warranted because incidence of HCC is increasing considerably in the United States, their dismal prognosis and the poor response of this cancer to treatment regimen currently available. This project focuses on the role of the loss of function of miR-122 in the development and progression of hepatocellular carcinoma (HCC), one of the most common human malignancies. Our previous studies have shown downregulation of miR-122 in HCCs of both human and rodent origin. Now we intend to show that the loss of function of this liver specific miR plays a causal role in the initiation and progression of HCC. If this is the case, these studies will open the way to the development of novel miR-122 therapy for this incurable disease. Thus, establishing role of miR-122 mimetic in an animal model (in preclinical trial) would be a major milestone in the treatment of this deadly disease in the near future.