沉默原癌基因Bmi-1的microRNAs在原发性肝癌中的功能及分子机制研究

Aim.The aim of this project is to identify the functions of the miR-218 in hepatocellular carcinoma (HCC) and the potential clinical correlations between miR-218 expression, Bim-1 expression and the clinical-pathological and prognostic parameters in a large series of HCC samples so as to provide the basis for the clinical application of miR-218..Project Background.MicroRNAs are the family of small non-coding single-stranded RNAs which repress gene regulation at the post-transcriptional expression level by base pairing to the 3' untranslated regions of target mRNA. These molecules play pivotal roles in various biological processes including differentiation, apoptosis, proliferation, and the maintenance of cell and tissue identity. The dysregulated expression of miRNAs has been linked to cancer and other diseases. Last year, we published the liver specific miR-122 suppressed HCC cell proliferation and induced apoptosis. And recently we reported that a primate specific miRNA-637 inhibit tumorigenesis in HCC through disrupting the Stat3 signaling. .Bmi-1, a polycomb gene family member, plays an important role in cell cycle regulation and cell senescence. Recently, numerous studies have demonstrated that Bmi-1 is involved in the carcinogenesis. Our lab justly reported that Bmi-1 promoted the invasion and metastasis of human breast cancer and predicted poor survival..Brief Project Description.In this proposal, we will investigate the functions of the conserved microRNAs, miR-218, targeting Bmi-1 in carcinogenesis of HCC. Bioinformatic analysis suggests that 6 miRNAs directly target oncogene Bmi-1. And our preliminary data indicated that Bmi-1 expression was up-regulated in a panel of HCC cell lines. Moreover, the expressions of these predicted miRNAs in HCC cell lines were assayed and miR-218 was the most significantly down-regulated miRNAs. Therefore, we hypothesize that miR-218 may play a role in carcinogenesis through directly targeting Bmi-1. In order to identify that Bmi-1 is a direct target of miR-218, 3'UTR of Bmi-1 was predicted to have two binding sequences which perfectly match the miR-218 seeds. Moreover, miR-218 was found to inhibit the proliferation of HCC cells. These results further support the hypothesis that miR-218 may act as a "suppressor" in HCC. .Significance of Project.Hepatocellular carcinoma (HCC) is a global public health problem that accounts for approximately 500,000 deaths annually. Considering the therapeutic options remains limited, the novel therapeutic targets and drugs are urgently needed. The present proposal will: (1) characterize the functions of miR-218 targeting Bmi-1 in HCC; and (2) investigate the potential clinical correlation between miR-218, Bmi-1 expression and the clinical-pathological and prognostic parameters in a large series of HCC tissue samples. The results from this project will provide new insights into the pathogenesis of HCC and explore a better molecular target for HCC diagnosis and therapy.

原癌基因Bmi-1参与了多种肿瘤的形成和发展。已有研究表明多个miRNAs可调控其表达从而起到抑癌的功能。但是，关于miRNAs调控Bmi-1表达从而影响肝癌的形成目前尚未见报道。前期实验证明了Bmi-1在肝癌细胞中表达明显升高，并且生物信息学预测出6个miRNA能较好的靶标该基因。进一步的实验显示，miR-218在肝癌细胞中表达被下调最为显著，并且它与Bmi-1的3'非翻译区有着较好的结合位点。由此，我们提出了miR-218调控肝癌的可能性，其分子机制可能是通过沉默Bmi-1的表达来实现的。本项目拟采用慢病毒表达技术以及荧光报告载体和裸鼠肝原位肿瘤模型，旨在获得miR-218靶标Bmi-1并调控肝癌的直接证据。并通过蛋白组学研究确定miR-218调控的下游靶标分子，从而深入阐明miR-218调控肝癌形成的分子机制，为确立miR-218作为肝癌治疗的分子新靶标提供更充分的科学依据。

肝癌是指发生于肝脏的恶性肿瘤，是临床上最常见的恶性肿瘤之一，根据最新统计，全世界每年新发肝癌患者约六十万，居恶性肿瘤的第五位。原发性肝癌按细胞分型可分为肝细胞型肝癌、胆管细胞型肝癌及混合型肝癌。按肿瘤的形态可分为结节型、巨块型和弥漫型。原发性肝癌在我国属于高发病，一般男性多于女性。中国是乙肝大国，我国的肝癌多在乙肝肝硬化的基础上发展而来。丙肝病人也正在逐年增加，它的后期也会发展成为肝癌。目前我国发病人数约占全球肝癌病人的55%。因此，肝癌已经成为严重威胁我国人们健康和生命的一大杀手，其危险性不容小视。近半个世纪以来，随着生物医学研究的飞速发展，肝癌的研究也取得了可喜的进展。目前肝癌的主要治疗手段是手术、介入栓塞化疗及肝移植等，但是术后并发症及高昂的医疗费用让很多患者望而止步。因此，寻找便捷有效的肝癌早期诊断及治疗的靶标仍是一个迫切需要解决的问题。..microRNA（miRNA）是近年来研究的一个热点，它是一类内源性的非编码的微小RNA，广泛存在于动物、植物以及病毒中。miRNA通过转录后水平调控靶标基因的表达，从而广泛的参与个体发育、器官形成、物质代谢等生理过程，并且与肿瘤的发生、病毒复制等病理过程有密切的联系。到目前为止，已经发现多个miRNAs在肝癌组织中异常表达，并且参与了肿瘤的形成。例如，miR-101，miR-29等在肝癌中表达被下调，他们可作为抑制子(suppressor)在肝癌形成中起到抑制作用。而相反的，miR-18a和miR-221/222作为原癌基因能促进肝癌的形成。..在本项目的研究中，我们发现miR-218在肝癌多个细胞株及50对组织样本中表达明显下降。体外细胞实验表明，miR-218高表达抑制肝癌细胞的增殖，引起肝癌细胞周期抑制，并且体内动物实验结果表明miR-218过表达抑制了肝癌细胞的形成。因此，miR-218在肝癌形成中作为一个tumor suppressor，其过表达抑制了肿瘤的形成的分子机制是通过miR-218直接靶标原癌基因Bmi-1从而激活了P14和P16这两条信号通路来发挥抑癌功能。这些研究结果表明miR-218可能会作为一个有希望的分子治疗靶标，为肝癌的治疗提供一个新的治疗靶点。