肝细胞癌1ncRNA表达谱研究

【作者】 刘武韬；

【导师】 黄洁夫；

【作者基本信息】 北京协和医学院 ， 外科学， 2014， 博士

【摘要】 研究背景肝细胞癌在全世界恶性肿瘤中其发病率和死亡率分别排在第六位和第三位。我国的新发肝细胞癌病例数占全世界所有新发病例数的55%。肝细胞癌与慢性乙型肝炎病毒感染密切相关,50%的肝细胞癌病例是由HBV感染导致。五年生存率只有5～6%,预后极差,造成严重的经济损失和沉重的社会负担。从正常的肝细胞演变为肝癌细胞直至发生最后的转移需经历多个病理阶段、发生多种分子事件改变。因此,阐明肝细胞癌的临床病理特征与其分子改变之间的关系,以发展出新的诊断和治疗策略,改善患者的预后,就成为当前迫切的需求和巨大的挑战。人类的基因组的组成除了编码蛋白质的组分,还包括数量远大于编码组分的非编码组分（超过97%）这些组分在疾病过程中也起着至关重要的作用。长链非编码RNA （long non-coding RNAs, lncRNAs）为数众多,但目前相关研究还非常缺乏。已有证据表明lncRNA在多种生理过程中发挥着极其关键而多样的调节功能,与包括癌症在内的多种致命性的人类疾病有密切关联。但是,有关lncRNA在肝细胞癌中所起的作用的研究还处于起步阶段,对其在肝细胞癌中的临床意义还知之甚少。研究目的发现肝细胞癌特异lncRNA表达谱。探索这些特异表达的lncRNA作为肝细胞癌诊断标记物的可行性。研究方法研究所用的病例都来自北京协和医院肝脏外科,时间跨度从2011年3月到2013年7月。选择了29例经病理学检查证实的肝细胞癌患者。所有受试者都接受了肝脏切除术,并在手术后立刻采集了组织样本。提取总RNA。首先利用人类lncRNA芯片检测了肝细胞癌组织和癌旁非肿瘤肝组织之间的lncRNA差异表达谱,然后选择其中5个lncRNA用qRT-PCR方法进行了定量验证。研究结果人类lncRNA芯片检测发现659个lncRNA在肝细胞癌组织和癌旁非肿瘤肝组织之间存在表达水平差异。从中选择的5个lncRNA （TCONS₀0018278, AK093543, D16366, ENST00000501583, NR₀02819（MALAT1））进行定量验证发现,与癌旁非肿瘤肝组织相比,其中TCONS₀0018278, AK093543, D16366, ENST00000501583在肝细胞癌组织中的表达水平出现显著下调（P=0.012,0.045,0.000and0.000）,而NR₀02819（MALAT1）在两种组织中的表达水平则没有显著性差异（P=0.114）。研究结论新一代的人类lncRNA芯片检测技术为lncRNA表达谱研究提供了良好的平台。肝细胞癌组织和癌旁非肿瘤肝组织之间存在大量的差异表达的lncRNA。lncRNA TCONS₀0018278,AK093543,D16366,ENST00000501583在肝细胞癌组织中的表达水平存在显著下调,为肝细胞癌的早期诊断和治疗提供新的思路。lncRNA MALAT1在肝细胞癌中的表达及意义还需要进一步评价和研究。更多还原

【Abstract】 BackgroundHepatocellular carcinoma （HCC） is the sixth most prevalent neoplasm and the third leading cause of cancer-related death. Especially, the new cases of HCC in China account for almost55%of all patients worldwide. With a dismal5-year survival rate of approximately5-6%, HCC is still one of the cancers with the worst prognosis and causes enormous social cost and burden. The carcinogenic process of HCC involves synergetic effects of multiple genetic and environmental factors, requires various pathologic stages and engages a number of molecular events. There is a great need for elucidating the relationships between clinicopathological features and molecular changes in HCC to develop new diagnosis and treatment strategies and improve the prognosis of diagnosed patients.In recent years, long non-coding RNAs （lncRNAs） have been shown to possess prominent and diverse regulatory functions in cancer processes. However, the roles of lncRNAs in hepatocellular carcinoma （HCC） and their clinical significances remain largely unknown.ObjectiveThe aim of this study was to exploit human lncRNA microarray for comprehensive and quantitative analyses of lncRNAs expression profiles in paired HCC tissues and adjacent non-tumor （NT） liver tissues and to evaluate the feasibility of using tissue lncRNAs as novel biomarker for the detection of HCC.MethodsThis was a retrospective laboratory study in a tertiary-referral university hospital in Beijing, PR China, between March2011and July2013. Twenty-nine subjects with histologically proven HCC were enrolled in this study. Liver resection was performed for each patient, and tissue samples were collected immediately after operation. Microarray-based lncRNAs expression profiling was used to identify differentially expressed lncRNAs between HCC tissues and adjacent non-tumor （NT） liver tissues, and quantification of selected lncRNAs was performed using quantitative real-time polymerase chain reaction （qRT-PCR）.Results659lncRNAs were differentially expressed between HCC tissues and NT tissues, of which five lncRNAs （TCONS₀0018278, AK093543, D16366, ENST00000501583, NR₀02819（MALAT1）） were selected for validation. Four of them were significantly down-regulated in HCC tissues compared with NT tissues （P=0.012,0.045,0.000and0.000, respectively）, and the expression level of MALAT1showed no significant difference （P=0.114）. ConclusionThis study reveals a set of lncRNAs differentially expressed in HCC tissues and provides useful information for exploring potential therapeutic targets and diagnostic biomarkers of this malignance. TCONS₀0018278, AK093543, D16366and ENST00000501583are down-regulated in HCC tissues and have the potential to be valuable biomarkers for diagnosing HCC. Further validations of the role of MALAT1in HCC are necessary.更多还原