【作者】 程恒辉；

【导师】 阮秋蓉； 王国平；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2013， 博士

【摘要】 肝细胞癌(Hepatocellularcarcinoma,HCC)是世界范围内常见的恶性肿瘤,但是目前为止除手术切除外,缺乏有效的防治手段。而且HCC的早期诊断率、早期发现率很低,临床上大多数患者发现时已处于疾病的中晚期,失去了早期治疗的机会。因此,如何对HCC有效的治疗和尽早的预防一直以来都是研究的热点与难题。肝纤维化是慢性肝病的病理基础,而HCC往往在慢性肝病的基础上发展而来,因此肝纤维化也是HCC的重要病理基础。HCC及肝硬化均是肝脏病变进展到不可逆阶段,但是肝纤维化阶段是可逆的,如果有效阻断甚至逆转肝纤维化,则慢性肝病、肝硬化及HCC便可获得良好的预防效果。microRNA在肿瘤的发生发展中有着重要作用,超过1/3的人类蛋白编码基因均受microRNA的调控。并且已有的研究显示,microRNA能够反映出疾病发生发展密切相关的分子本质,能够运用于疾病的发病机制研究、诊断、分子分型、治疗及预后的判断,具有极大的研究潜力。同时运用microRNA与mRNA对接研究的方法可以大大提高microRNA靶基因预测的准确性,便于寻找目的靶基因。本课题分别从HCC的早期预防——探讨肝纤维化发病机制的角度以及探索HCC新的治疗方法角度,运用microRNA或mRNA表达谱筛选的方法,对差异表达的RNA进行生物信息学分析,为HCC的早期预防及有效治疗提供新的依据。本研究分二个部分。第一部分:肝窦内皮细胞毛细血管化相关的microRNA及靶基因研究目的:筛选出在肝窦内皮细胞(LSEC)毛细血管化过程中差异表达的mRNA和microRNA,并进行生物信息学分析,根据分析结果挑选靶基因进行组织学验证,为深入探讨肝窦内皮毛细血管化的关键基因提供前期依据。方法:体外持续培养人原代肝窦内皮细胞(HHSEC),在扫描电镜下观察培养过程中窗孔结构的维持情况。根据窗孔的有无进行分组,随后提取mRNA进行mRNA及microRNA表达谱芯片研究,将研究结果进行生物信息学分析及对接研究,挑选差异明显的靶基因进行临床大样本验证。结果:通过体外培养HHSEC的方法可以观察到窗孔结构由有至无的变化,我们提取相应mRNA进行mRNA和microRNA表达谱芯片。mRNA表达谱芯片结果显示,1639个基因出现表达上调、1608个基因表达出现下调。其中,有532个mRNA差异显著。microRNA表达谱芯片筛选出了与HHSEC毛细血管化相关,P<0.05,差异显著的microRNA共19个,其中表达上调的microRNA共7个,表达下调的microRNA共12个,并通过生物信息学分析方法获得了 10109个靶基因信息。进一步将mRNA与microRNA研究结果进行对接分析,结果获得253个共同的靶基因。根据这253个靶基因差异倍数大小挑选出差异显著的一个——CALM2进行临床组织样本免疫组化验证,结果显示CALM2在LSEC的表达水平与肝纤维化的程度成正比,证实该靶基因与肝纤维化有关。结论:结合HHSEC mRNA及microRNA表达谱芯片筛选及生物学分析结果获得与HHSEC毛细血管化相关的靶基因,并证实该靶基因的蛋白表达与肝纤维化程度成正比,为深入探讨肝纤维化发病机制及逆转措施提供了有力的实验依据。第二部分:调控肝细胞癌中Glypican-3表达的microRNA研究目的:筛选出HCC中与Glypican-3(GPC3)表达相关的microRNA,并进行生物信息学分析,根据分析结果挑选差异显著的microRNA进行组织学验证,为深入探讨GPC3在HCC表达的发生机制提供RNA依据。方法:将有完整临床病理学资料的HCC样本进行GPC3组织学免疫组化及mRNA检测,并据此分为GPC3阳性组和GPC3阴性组,进行microRNA表达谱芯片研究。将研究结果进行生物信息学分析,挑选差异明显的microRNA进行临床扩大样本验证。结果:根据GPC3表达情况进行分组后,microRNA表达谱芯片筛选出信号值>500共有25个差异表达的microRNA,其中10个microRNA表达上调,15个microRNA表达下调。随后挑选出5个microRNA进一步qPCR扩大样本量验证,成功筛选出了 4个差异表达microRNA(miR-99a-5p,miR-125b-5p,miR-376c-3p,miR-17-5p),并通过生物信息学分析方法获得了相关的GO和信号通路信息。结论:运用microRNA表达谱芯片和qPCR扩大样本量验证的方法成功筛选出了 HCC中GPC3相关的microRNA,并通过生物信息学分析为后续实验研究提供可靠的实验数据。结论:肝纤维化及HCC的形成过程中有大量microRNA和靶基因的共同参与,我们通过microRNA或mRNA表达谱分析,筛选出了在各自过程中差异显著的microRNA及mRNA,并利用生物信息学分析方法获得了相关靶基因和信号通路信息,同时进行临床验证,为深入研究提供前期基础。更多还原

【Abstract】 Hepatocellular carcinoma(HCC)is a common malignant tumor in worldwide,but so far,there still have no effective measures beside surgery.And low early diagnosis and detection rate of HCC made the loss of early treatment clinically for the majority of patients were found with advanced disease.So,seeking new effective treatment and early prevention of HCC are always been the research focus and important problems.Liver fibrosis is the pathological basis of chronic liver disease,and HCC often develops on the basis of patients with chronic liver disease.Therefore,liver fibrosis is also an important pathology basis of HCC.HCC and liver cirrhosis are all progressed to an irreversible stage,but liver fibrosis is reversible.So,if we can effectively block or even reverse fibrosis,good preventive effect of the chronic liver disease,cirrhosis and HCC could be obtained.MicroRNA plays an important role in tumor development,more than 1/3 of the human protein-coding genes are subject to microRNA regulation.As research reported,microRNA can reflect the molecule nature closely related to the development of the disease,and has a great research potential role in beening applied to research the disease etiology,diagnosis,molecular typing,treatment and prognosis.Meanwhile,microRNA and mRNA crossing study can greatly improve the target gene prediction accuracy of microRNA and make finding target gene more easily.This research aiming to find new early prevention and treatment of HCC from investigating the pathogenesis of liver fibrosis and HCC,using microRNA and mRNA expression profiling screening method to analyze the differential expression of RNA bioinformatics,provides new basis for HCC early prevention and effective treatment.This study is composed of two parts.Part I:MicroRNAs and target genes screening research in intervention of the liver sinusoidal endothelial cell capillarisationObjective:To filter out the differentially expressed mRNA and microRNA in liver sinusoidal endothelial cell(LSEC)capillarisation,do bioinformational analysis,and according to the screening results,select target genes to do histological verification,providing initial basis on researching key genes in LSEC capillarisation.Methods:Cultured human hepatic sinusoidal endothelial cells(HHSEC)in vitro,investigated fenestration in scanning electron microscopy and grouped.Extracted mRNA for mRNA and microRNA microarray test,the results of the study did bioinformational and crossing analysis studies,evidently different target gene would be selected for clinical validation.Results:Fenestration could be observed gradually disappearing in in vitro cultured HHSEC.Extracted RNA for mRNA and microRNA microarray.mRNA microarray results showed that 1639 genes upregulated and 1,608 genes downregulated.532 out of these mRNA had significantly difference.MicroRNA microarray screening found 19 microRNAs had significantly relationship with HHSEC capillarisation(P<0.05),including 7 microRNAs upregulation,12 microRNAs downregulation.Bioinformational analysis methods obtained 10 109 target genes information.Further studies of mRNA and microRNA crossing analysis counted 253 target genes and chosen CALM2,one of the most obviously genes for a clinical histological verification.The results showed CALM2 expression was proportionate to liver fibrosis that confirmed the relationship of the target gene and liver fibrosis.Conclusion:Combining HHSEC mRNA and microRNA microarray screening and biological analysis results obtained target genes which confirmed with relationship of HHSEC capillarisation and liver fibrosis.This would provide a strong experimental basis for further investigation of the pathogenesis and reversal measurement of liver fibrosis.Part II:MicroRNAs regulated Glypican-3 expression in hepatocellular carcinomaObjective:To filter out Glypican-3(GPC3)expression associated microRNA in HCC,and do bioinformational analysis,then according to screening results select the most different microRNA for histological verification,giving RNA basis on GPC3 expression mechanism in HCC.Methods:GPC3 immunohistochemical staining and mRNA level in HCC samples with complete clinical pathological data were carried out and divided into GPC3 positive or negative group according to detected results.Further microRNA microarray screening was processed and bioinformatiinal analysis were done.Selected the obviously different microRNAs to validate in expanded sample.Results:MicroRNA microarray of different GPC3 groups screened out 25 different microRNAs(signal value>500),of which 10 were up-regulated and 15 were down-regulated.5 microRNAs were chosen for qPCR validation on expanded samples and successfully screened out 4 differentially expressed microRNA(miR-99a-5p,miR-125b-5p,miR-376c-3p,miR-17-5p),and obtained relevant information of GO and signaling pathways through biological information analysis.Conclusion:Using microRNA microarray and expanded sample qPCR validation method successfully screened out GPC3 associated microRNAs in HCC,and provided reliable experimental datas for subsequent experimental research by bioinformational analysis.Conclusion:A large numbers of target genes and microRNAs participate liver fibrosis and HCC formation.We screened out significantly different microRNAs or mRNAs by mRNA and microRNA expression profiling,and obtain the relevant target genes and signaling pathways information using bioinformational analysis methods,and validated on clinical expanded sample.This would provided initial basis for further research.更多还原