【作者】 肖奋强；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 普通外科学， 2014， 博士

【摘要】 背景：原发性肝细胞癌(Hepatocellular carcinoma, HCC)是全球患者死亡率居前的恶性肿瘤,也是我国常见的恶性肿瘤之一。与其它恶性肿瘤一样,肝癌的发生与发展是包括原癌基因激活或／和抑癌基因失活的多阶段多步骤的过程。HCC的发生与慢性肝炎及肝硬化密切相关,是一个逐步发展的渐进过程,并且HCC的发生与乙肝病毒(HBV)的感染有较高的关联性。由于我国约有1.2亿乙肝病毒携带者,占全球感染人数的1/3,如何有效地干预HCC的发生和治疗HCC患者成为了重大紧迫的问题。因此,寻找HCC的发病机制和发现有效的治疗靶点具有重要的意义。越来越多的研究结果证明,除了HCC内蛋白编码基因的变异,miRNA表达的失调也会促进HCC的发生。microRNA (miRNA)是近年来发现的一类长度仅为18-25个核苷酸的内源性非编码小RNA,成熟miRNA的5’非翻译区的2-7个核苷酸的“种子序列”可以与靶mRNA的3’-非翻译区互补结合,在转录后水平上抑制靶基因的表达。1993年,首个microRNA-lin4被发现,从此揭开了对microRNA研究的序幕。随后,许多实验室从线虫、果蝇、家鼠、拟南芥及人类等多种真核生物中发现了至少500种这类小分子RNA,其中在人类染色体上就有300余种,占人类基因的1-4%。miRNA的编码基因是目前最大的一类调节基因。它在细胞的生长、增殖、发育和凋亡过程中发挥着重要作用,并与许多肿瘤的发生、发展相关。rniRNA在肿瘤中发挥着相当于癌基因或抑癌基因的作用,调节着肿瘤细胞的多种重要的生物学行为。目前发现很多miRNAs在肝癌中异常表达,并在肝癌的发生发展中扮演着重要的角色。这些异常表达的miRNA包括,miR-1、miR-2、miR-221、miR-222、 miR-224, miR-301, let-7a, miR-101、miR-122、miR-125a、miR-139a、miR-143、 miR-195、miR-26, miR-29和miR-199a/b-3p等。这些miRNA的失调能够通过影响细胞的生长、凋亡.迁移和侵袭等生物学过程并影响HCC的发生和进展。但是,对于失调的miRNA如何参与HCC的发生和发展的机制尚有待于进一步深入研究,HCC中新的失调的miRNA及其功能的探索,将为HCC的治疗和预后判断提供新的理论基础。目的：本研究旨在寻找HCC中差异表达的miRNA,再用实时荧光定量PCR的方法验证差异表达的miR-503在肝癌组织及肝癌细胞系中的表达水平,并统计分析miR-503表达量与HCC患者临床病理参数和预后的关系。在此基础上进一步以获得性和缺失性功能实验明确miR-503在肝癌细胞中的生物学功能,分析并验证其发挥作用的直接靶基因,探索其在肿瘤发生发展中可能的分子机制。方法：1.利用microRNA实时定量PCR芯片技术高通量筛选肝癌细胞株和正常永生化肝脏细胞差异表达miRNA(由上海康成生物工程有限公司协助完成)。2.进一步用实时荧光定量PCR法验证明显差异表达的miR-503在肝癌组织及肝癌细胞系中的表达情况。收集125例肝癌患者肿瘤及周围非瘤组织手术标本,7株肝癌细胞系及2株永生化肝细胞系,经提取RNA,反转录后得到cDNA,运用实时荧光定量PCR技术检测miR-503的表达水平。3.根据miR-503在肝癌组织中的表达水平,将HCC患者分为miR-503高表达组和低表达组,进而分析miR-503表达量与HCC患者临床病理参数及预后之间的关系。4.通过体外转染niR-503mimic和inhibitor的方式来进行获得性和缺失性功能实验。以CCK-8、克隆形成、裸鼠体内成瘤实验、划痕实验、流式细胞周期检测、Edu实验等检测miR-503对细胞增殖与迁移的影响。5.利用在线数据库预测miR-503可能的靶基因,并通过双荧光素酶报告系统、Western blot等技术鉴定miR-503的靶基因。并进一步探讨miR-503参与细胞周期调控的分子机制。结果：1. microRNA定量PCR芯片结果提示：相对于正常肝脏细胞,miR-503, miR-218, miR-424, miR-30e, miR-147, miR-450a, miR-411等在HCC细胞株中的表达下调,选择其中表达明显下调的miR-503做进一步研究。2.分别检测了125例肝癌组织及配对的癌旁组织、7株肝癌细胞系及一株永生化肝细胞系L-02中miR-503的表达水平,发现在大部分肝癌组织和肝癌细胞系中miR-503表达水平降低,差异有显著性。3.miR-503表达水平与甲胎蛋白(P=0.015)、肿瘤病理学分级(P=0.019)、TNM分期(P=0.012)及门静脉癌栓(P=0.029)密切相关,但与年龄、性别、肿瘤大小、肿瘤数量无关,miR-503的低表达与HCC患者更短的总体生存期相关,提示miR-503可以作为一个判断HCC患者预后的独立预测因子。4.体内和体外功能研究结果显示：CCK-8实验、Edu实验、克隆形成实验及裸鼠皮下成瘤实验证实miR-503抑制了肝癌细胞的体内体外增殖能力；划痕实验提示miR-503过表达抑制了LM3细胞的迁移。流式细胞周期检测发现miR-503可以诱导肝癌细胞发生G1期阻滞。5.靶基因预测分析结果与细胞表型变化相结合,确定细胞周期相关分子cyclin D3和E2F3为候选靶基因。Western blot表明：LM3细胞中过表达miR-503明显下调Cyclin D3和E2F3蛋白的表达,而其mRNA水平无明显变化,提示miR-503可能通过抑制靶基因的翻译发挥作用。6.构建pmirGLO-cyclin D3-UTR和pmirGLO-E2F3-UTR野生型及突变型荧光素酶报告载体,将miR-503mimic与野生型荧光素酶报告载体共转染293T细胞后,荧光素酶报告基因活性明显降低,而共转染突变型荧光素酶报告载体后荧光素酶报告基因活性无明显影响。表明miR-503过表达可直接作用于cyclin D3和E2F33’UTR的靶序列,下调靶基因的表达。7.miR-503过表达显著下调Rb-E2F信号通路中关键分子CDK4, CDK6,磷酸化RB及E2F3下游基因cyclin A和Cdc2蛋白水平的表达,表明miR-503通过Rb-E2F信号通路抑制了肝癌细胞周期G1/S转换。结论：1.miR-503在HCC中表达水平频频下调,暗示其表达异常可能与HCC的发生发展密切相关。2.HCC组织中miR-503的表达水平与HCC患者的临床病理参数及总体生存期相关,暗示miR-503的表达量可以作为判断HCC患者预后的一个潜在的预测指标。3.miR-503诱导肝癌细胞发生细胞周期G1期阻滞及显著抑制细胞增殖。4. cyclin D3和E2F3被鉴定为miR-503的直接功能靶标。5.miR-503通过Rb-E2F信号通路参与肝癌细胞周期调控。更多还原

【Abstract】 BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The development and progression of HCC is typical of a multistage process, which is believed to involve the deregulation of genes that are critical to cellular processes, such as cell cycle control, cell growth, apoptosis and cell migration and spreading. In the past decades, studies have focused on investigating the genes and proteins underlying the development and progression of HCC. Recently, an increasing number of reports have described a new class of small regulatory RNA molecules termed microRNAs (miRNAs) that are implicated in HCC progression.MicroRNAs (miRNAs) belong to a large family of non-coding, single-stranded RNAs,18-25nucleotides long. There is growing evidence that miRNAs regulate the expression of protein-coding genes, either at the level of messenger RNA degradation or translation, by pairing with their target mRNAs3’UTR. In1993, the first miRNA lin-4was discovered. They are involved in many biological processes including cell cycle regulation, development, differentiation, metabolism, and ageing. miRNA dysregulation has been observed in many human malignancies and there is some evidence for their involvement in the progression of tumors, either as oncogenes or tumor suppressors. However, their exact biological role in HCC remains unclear. Further research is still needed to explore new deregulated miRNA and its functions, which provide new ideas for the treatment and prognosis of HCC.AimsThis study aimed to find differentially expressed miRNA in HCC, and then quantitative PCR was used to validate the miRNA expression level in HCC tissues and cell lines. Based on this we further investigated the biological function of miR-503in HCC cells by using gain-of and loss of function experiments, analyzed and verified its target genes and explored the possible molecular mechanisms of HCC in development and progress.Methods1. MicroRNA real-time quantitative PCR chips was used to explore differential expression miRNA in HCC cell lines and normal immortalized liver cells.2. We verified the expression of miR-503in125paired samples of clinical HCC tumor, adjacent normal liver tissues and7HCC cell lines using quantitative R-T PCR analysis and then investigated the correlation between clinicopathologic factors and miR-503expression.3. We conducted gain-of and loss of function analysis by transfecting HCC cell lines with chemically synthesized miR-503mimics or inhibitor. Methods including CCK-8, colony formation assays, Edu assays, tumor formation in nude mice and flow cytometry were used to measure cell proliferation and cell cycle.4. We investigated the direct target genes of miR-503involved in cell cycle arrest by a dual-luciferase reporter system, Western blot and qPCR.Results1MicroRNA array showed that miR-503was significantly downregulated in HCC cell line. 2. Further study verified that significant downregulation of miR-503was observed in HCC tissues compared with their adjacent non-tumor tissues. Furthermore, the expression of miR-503was noticeably reduced in five of seven (71.4%) HCC cell lines examined compared with normal liver cell line L023. Lower miR-503expression in HCC tissues significantly correlated with shorter survival of HCC patients, and miR-503was identified to be an independent predictor for prognosis of HCC patients.4. Restoration of miR-503dramatically suppressed HCC cell growth in vitro and in vivo by inducing G1phase cell cycle arrest.5. We confirm cyclin D3and E2F3are direct functional targets of miR-503by using a dual-luciferase reporter system.ConclusionsIn conclusion, we report the altered miR-503expression pattern in HCC, investigate the potential role of miR-503in tumorigenesis, and demonstrate that cyclin D3and E2F3are direct functional targets of miR-503. Our data demonstrate an important role for miR-503in the molecular etiology of cancer and suggest its potential application in cancer therapy.更多还原