【作者】 徐力致；

【导师】 王亚平；

【作者基本信息】 南京大学 ， 内科学， 2011， 博士

【摘要】 肝癌是人类常见的、高恶性程度的肿瘤之一,位居全球恶性肿瘤发病率的第5位、肿瘤死因的第3位。肝癌的发病率以亚洲和非洲国家为高,在我国更是成为恶性肿瘤的第2位杀手。引起肝癌发病的主要因素包括肝炎病毒(HBV、 HCV)感染、食品黄曲霉素(aflatoxin, AF)污染、饮水微囊藻毒素(Microcystin,MC)污染以及遗传因素等。尽管目前提出了多种理论解释肝癌的发生和发展,但是这种疾病的确切发生机制仍不清楚。内源性表达的小分子非编码RNA,称为微小RNA(microRNA, miRNA)是近年发现的一类核酸组分,广泛参与生物基因的表达调控,人类基因组中约1/3基因的表达可看到miRNA调控的存在,并在细胞生长、分化、凋亡及肿瘤发生中发挥了关键作用。研究显示miRNA的表达异常贯穿于肝癌发生的整个过程,某些关键miRNA表达水平的失衡是肝癌发生和演进的重要分子事件。鉴于miRNA变化具有的前瞻性,即在肿瘤形成之前其已呈现可检出的表达异常。研究miRNA与肝细胞转化、成瘤演进过程中的变化,对于临床的早期诊断与治疗、以及监测环境微量污染物潜在致癌作用具有重要意义。本研究探讨了miRNA在肝癌病人组织和淡水污染物——微囊藻毒素诱导肝细胞恶性转化中的差异表达,筛选与肝癌相关的miRNA作为生物标记物,为临床诊断与治疗,以及环境毒物致肝癌发生提供科学依据。第一部分：miRNA在肝细胞性肝癌临床组织样本中差异表达的检出及其生物学的研究目的探寻肝细胞性肝癌(HCC)中异常表达的特征性miRNA,分析其引发HCC的可能机制及其作为临床检测生物标记物的可能性。二、方法1、利用MicroRNA microarray技术,分析比较临床肝癌组织与其同源癌旁组织miRNA差异表达谱。2、利用荧光定量RT-PCR技术比较miR-491-3p和miR-875-3p在85例HCC组织中的差异表达。3、构建miR-491和miR-875前体质粒,转染入HepG2细胞,流式细胞技术分析其对细胞周期的影响。三、结果1、四例HCC组织miRNA差异表达谱显示,在癌组织中发生明显上调miRNA22种,下调miRNA20种。2、荧光定量RT-PCR技术筛检发现,miR-491-3p在HCC病人组织中下调比例为41.2%,在非肝硬化及非静脉侵袭的癌组织中下调更为明显,且与细胞分化程度相关。miR-875-3p在HCC病人组织中下调比例为60%,在非肝硬化及非包膜侵袭的癌组织中下调更为明显,且与细胞分化程度相关。3、流式细胞检测显示,miR-491-3p可以引起细胞的凋亡增加,而miR-875-3p则引起细胞阻滞于G0/G1期。四、结论HCC中普遍存在miRNAs的表达异常。其中miR-491-3p和niR-875-3p在HCC中明显下调,且与肝癌的临床特征相关联,可能成为发展肝癌临床诊断和治疗技术的分子靶点。第二部分：miRNA在微囊藻毒素-LR诱导肝癌发生中的作用一、目的建立MC-LR诱导肝细胞恶性转化模型,分析其miRNA的差异表达,并探讨miRNA作为MC-LR诱导肝癌发生监测分子靶标的可能性及意义。二、方法1、MC-LR连续刺激WRL-68细胞后,采用MTT法检测细胞增殖及血清依赖情况,流式细胞技术观察细胞周期变化,裸鼠成瘤实验显示细胞恶性程度。2、利用MicroRNA microarray技术MC-LR诱导恶性转化细胞进行miRNA差异表达谱分析。3、小鼠MC-LR饮水喂养,通过血清ALT、 AST和组织切片分析小鼠肝损伤状态。4、荧光定量RT-PCR技术测定小鼠肝脏miR-21、 miR-122、 miR-195、 miR-221、 miR-222、 miR-338-5p和miR-491-3p的差异表达。三、结果1、 MC-LR连续刺激WRL-68细胞25代后,细胞增殖增强、血清依赖性下降、出现多倍体峰,且能在裸鼠体内成瘤,证实MC-LR可以导致体外细胞恶性转化。2、MC-LR导致体外细胞恶性转化miRNA差异表达谱显示,有126个明显改变(2倍以上变化),其中78种miRNA表达上调,48种miRNA表达下调。3、MC-LR80ug/L组连续饮水9个月,小鼠肝细胞双核明显增加,提示MC-LR可引起小鼠肝细胞基因组不稳定,具有发生恶性转化的潜能。4、荧光定量RT-PCR技术测定显示,小鼠肝脏慢性MC-LR毒染过程中,miR-21、 miR-221和miR-222表达上调,miR-122、 miR-338-5p和niR-491-3p表达下调,miR-195在40ug/L组表达上调,而80ug/L组表达下调。四、结论本研究显示,MC-LR诱导的体内外肝细胞恶性转化过程中均存在miRNA的差异表达。miRNA的特征性表达可能成为环境污染物的分子检测指标。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is the fifth most common malignant cancer and the third leading cause of death of cancer-related death. Although most cases occur in Asia and Africa, the incidence has been steadily increasing in the west over the last20years. The major etiologies of HCC include hepatitis B or C virus infection, aflatoxin or water pollution and hereditary factor. It has been known that the hepatocarcinogenesis involves a complex, multistep process, which is linked tightly to chronic liver damage. A variety of molecular alterations that can result in the loss of cell-cycle checkpoints, resistance to apoptosis or activation of oncogenic pathways occurred at the initiation stage and in the development of liver cancer. However, neither of these molecular alterations has steadily been revealed in hepatocellular carcinomas. Recent studies suggested that miRNA expression alteration could play an important role in pathogenesis of HCC.MiRNAs are small non-coding RNAs of20-22nucleotides, which control a wide range of biological processes including developmental timing, cell proliferation, differentiation, apoptosis, and metabolism, etc. MiRNAs interact with target mRNAs at specific sites within their3’-untranslated regions (UTRs) to inhibit protein translation or induce the target mRNAs cleavage. More than60%of human protein-coding genes are predicted to contain miRNAs binding sites and therefore subject to miRNAs regulation. Furthermore, aberrant expression of miRNAs has been associated with various human diseases. A considerable body of evidence support that miRNAs can control the regulation of cancer development, and also represented in the environmental pollutant-and toxicant-induced carcinogenesis. All of the investigated human cancers, including HCC, are characterized by globally abnormal miRNA expression patterns. Moreover, the association demonstrated between the characteristic miRNA expression patterns and cancer types suggests that different response of miRNAs is observed among cancers from different tissues of origin. The special changes of miRNAs expression in cells can stimulate a cascade of cancer development. Therefore, the assay of miRNAs expression alteration in early stages of cancer promises an insight into the underlying mechanisms of carcinogenesis.In the present study, we sought to explore the altered expression of miRNAs underscored in HCC patients and the MC-LR-induced hepatocarcinogenesis. We consider that these results will promise to identify HCC and MC-LR-related miRNA alterations in tumorigenesis, which could be taken as biomarkers for using diagnosis in HCC or in water environmental monitoring, even for the development of miRNA-based prevention and treatment of hepatocellular carcinoma.Part Ⅰ miRNA profiling in hepatocellular carcinoma is associated with clinical featuresObjectiveIn this present study, we examined the expression profiles of miRNA in4pairs of HCC tumorous tissue and adjacent non-tumorous tissue by microRNA microarray method and then identified differentially expressed miR-491-5p and miR-875-5p in85patients to analysis the correlation between HCC and their clinic phenotype. Different miRNA expression could have relevance to the clinical behavior HCC and the miRNA expression may prognosticate disease outcome in HCC.Methods1. MicroRNA microarray experimentation including labeling, hybridization, scanning, normalization and data analysis was conducted by Exiqon A/S Technology Platform (Vedbaeck, Denmark). 2. TaqMan microRNA real-time quantitative-PCR analysis85HCC tumorous tissue and adjacent non-tumorous tissue for miR-491-3p and miR-875-3p.3. The recombinant plasmids expressing miR-491-3p and miR-875-3p were obtained by transfection of pEGFP-C1-miR-491and pEGFP-C1-miR-875into HepG2cells. We used PI staining to determine the cell cycle.Result1. The first aim of the present study was to investigate whether aberrantly expressed miRNAs between HCC tissues (T) to their corresponding normal tissues (NT). We identified22miRNAs were significantly upregulated expressed and20miRNAs were significantly downregulated expressed in the HCC tissues (mean fold change>2or<0.5).2. MiRNA specific real-time RT-PCR was used to test miR-491-3p and miR-875-3p expression lever in85HCC tissues to their corresponding normal tissues. The relative expression lever of these miRNAs significantly differed in T and NT. MiR-491-3p (41.2%) and miR-875-3p (60%)(Wilcoxon signed-rank test) were down-regulated in T.3. Of all variables tested, the statistical analysis only revealed that the miR-491-3p and miR-875-3p expression lever is lower in non-cirrhosis compared with cirrhosis (p=0.017and p<0.001) and in poorly differentiation (p<0.001and p<0.001).4. The transfection of pEGFP-C1-miR-491cells represented a significant enhanced proportion of apoptosis in cell cycle analysis, and the transfection of pEGFP-C1-miR-875cells had a significant enhanced proportion of G0/G1phase and a reduced of G2/M and S phase.ConclusionIn the present study, we have shown for the first time that the expression of miR-491-3p and miR-875-3p were downregulated in HCC. Results reported here may provide a useful clue for the research into HCC. Further investigation is needed to expound the function of miR-491-3p and miR-875-3p in the HCC, which may lead to finding new methods to diagnose, treat and prevent HCC.Part II Alteration of microRNA Expression Profile Linked to Microcystin-LR-Induced TumorigenicityObjectiveMC-LR has been shown to be a cyclic heptapeptide that acts as a potent hepatotoxin and cancinogen. However, the mechanism for its carcinogenic action remains to be determind. In this work, we used MC-LR to induce the malignant transformation in vitro and in vivo, and analyzed the miRNA expressed alteration in the transformed cells for addressing the miRNA role in the process.Methods1. The cultured WRL-68cells were continuously exposed to low concentration (10μg/L) of MC-LR for25passages (25MC10). We tested for their growth kinetics, resistance to serum-induced terminal, cell cycle and tumorigenicity in nude mice to investigate the transformed cells.2. MicroRNA microarray experimentation including labeling, hybridization, scanning, normalization and data analysis was conducted by Exiqon A/S Technology Platform (Vedbaeck, Denmark).3. The mice were fed on commercial laboratory chow and give MC-LR water. Serum was separated by centrifugation, and serum AST and ALT activities were estimated. Liver sections were taken and fixed in4%neutral-buffered formalin and prepared for examination under a photomicroscope.4. TaqMan microRNA real-time quantitative RT-PCR analysis mice liver for miR-21、 miR-122、 miR-195、 miR-221、 miR-222、 miR-338-5p and miR-491-3p.Result1. The cultured WRL-68cells were continuously exposed to low concentration (lOμg/L) of MC-LR for25passages (25MC10). Compared to the mocked treated parental cells, the induced25MC10cells represented a higher growth rate, a resistance to serum-induced terminal differentiation, and the tumorigenicity phenotype in nude mice xenograft test.2. Array-based miRNA expression profiles showed that78of miRNAs were up-regualted and48were down-regualted respectively in the25MC10cells.3. While treatment of MC-LR80ug/L in9months, serum AST and ALT activities were remarkably increased. The histological pattern was significant increased of dicaryon in hepatoceyte, which showed MC-LR induced malignant transformation in vivo.4. MiRNA specific real-time quantitative RT-PCR was used to test. miR-21、 miR-221and miR-222were upregulated, and miR-122、. miR-338-5p and miR-491-3p were downregulated in MC-LR-treatment livers. miR-195were upregulated in40ug/L group, but downregulated in80ug/L group.ConclusionThese results suggest that chronic exposure to MC-LR can alternate the miRNA expression profile of hepatocarcinogenesis in vitro and in vivo. The characteristic miRNA alterations could be taken as the molecular targets for developing environment molecular inspection techniques.更多还原