【作者】 桂俊豪；

【导师】 田亚平；

【作者基本信息】 中国人民解放军军医进修学院 ， 临床检验诊断学， 2010， 博士

【摘要】 非编码RNA (non-coding RNA, ncRNA)是当前生命科学研究的前沿热点之一,其中,microRNAs (miRNAs)是一大类新型的、细胞内源性的单链非编码小RNA,由基因组中的miRNA基因(miRNAs genes)编码。成熟miRNAs的平均长度约22 nt,广泛分布于各种生命体,作为RNA诱导的沉默复合体(RNA-induced silencing complex, RISC)的核心功能要素之一,主要通过与其靶mRNA (messenger RNA, mRNA)的3’-UTR区(3’-untranslated region,3’-UTR)互补结合,进而抑制mRNA的翻译或诱导mRNA降解,发挥RNA介导的转录后基因沉默(post-transcriptional gene silencing, PTGS)效应,对细胞增殖、分化、凋亡等一系列生命现象发挥广泛的生物学调控作用。据生物信息学预测,人类基因组编码的miRNAs可能超过1000种。研究发现,在不同的生理病理状态下,组织、细胞的miRNAs表达谱具有独特的时空特异性和组织特异性,miRNAs异常表达调控与肿瘤等多种人类疾病的发生发展密切相关。最近研究显示,血浆或血清中的循环miRNAs (circulating miRNAs, cir-miRNAs)可能是一类具有潜在临床应用价值的新型生物标志物(biomarker)。业已表明,肿瘤、心血管病等疾患个体的血浆或血清中存在如miR-92和miR-1等一些异常表达的、可能有助于疾病诊断及预后评估的循环miRNAs。基于目前循环miRNAs的研究新进展,我们有理由推测：在正常机能状态下,机体内循环miRNAs的种类及其表达丰度可能处于一个相对稳定、平衡的状态,而机体内器官组织的功能、代谢异常或器质性病变等因素可能会直接或间接地破坏机体内循环miRNAs的稳态(homeostasis),表现为特定生理或疾病状态下某些循环miRNAs的表达水平的变化。乙型肝炎病毒(hepatitis B virus, HBV)感染、慢性乙型肝炎(chronic hepatitis B,CHB)(简称慢性乙肝)、肝纤维化(liver fibrosis,LF)、肝硬化(livercirrhosis, LC)和肝细胞癌(hepatocelluler carcinoma, HCC)等肝脏疾病(简称肝病)依旧严重危害人们身体健康,进一步发展更加特异、敏感的肝脏疾病生物标志物可以弥补ALT、AST、AFP等传统血清学标志物的不足,对于提高肝脏相关疾病的辅助诊断、评估及健康管理等具有十分重要的临床意义。因此,为探索循环miRNAs对于肝脏相关疾病辅助诊断的潜在意义,本论文主要进行了四个方面的工作：第一,建立利用实时定量PCR (real-time quantitative PCR, rt-qPCR)对临床血清标本中循环miRNAs进行定量测定的技术平台。第二,采用基于qPCR (quantitative PCR)的TaqMan低密度阵列(TaqMan low density array, TLDA)技术,对健康个体、肝细胞癌(HCC)和肝硬化(LC)患者血清参考标本中的循环miRNAs进行了初步分析,以期筛选潜在的肝脏疾病相关的循环miRNAs。第三,通过分析筛选结果,选择5种在HCC和LC血清中表达上调的、可能与肝脏疾病相关的miRNAs(即miR-146a,-215,-224,-573-3p和miR-885-5p)进行初步验证。第四,基于有关文献进行数据挖掘,分析miR-885-5p在不同人体组织中的表达及分布,进一步采用TargetScan等生物信息学软件对miR-885-5p的潜在靶基因进行预测和分析。主要获得以下四个方面的结果：第一,建立了循环miRNAs定量的技术路线。第二,验证了TLDA技术用于循环miRNAs筛选的可行性。第三,发现慢性乙型肝炎、肝硬化及肝细胞癌患者血清中miR-885-5p的表达丰度显著升高。ROC (receiver operating characteristic, ROC)分析显示,miR-885-5p用于鉴别肝脏相关疾病与健康个体时的曲线下面积(the area under curve,AUC)为0.904,特异性为79.17%,敏感性为90.53%。最后,基于文献分析,发现miR-885-5p在肝脏组织中具有较高的表达水平。生物信息学分析显示,在胆固醇逆向转运(cholesterol reverse transport, CRT)及磷脂等脂类代谢中具有重要功能的ATP结合盒转运体超家族成员ABCA1 (ATP-binding cassette sub-family A member 1, ABCA1)是miR-885-5p的潜在靶基因之一。本论文建立的技术平台可为循环miRNAs的实时定量提供参考,发现并初步验证了血清miR-885-5p可能是一种有助于肝脏疾病诊断的新型标志物。更多还原

【Abstract】 Non-coding RNA (ncRNA) including microRNAs (miRNAs) have currently become one of the frontiers and hotspots of life science. MiRNAs are a large class of single-strand endogenous non-coding small RNA of approximately 22 nucleotides (nt) in length that encoded by miRNAs genes in genome and found in a wide varity of organisms. In recent years, it was uncovered that mature miRNAs function as one of the key components of RNA-induced silencing complex (RISC) to perform their post-trancriptional gene silencing (PTGS) effects, by binding to specific sites in the 3’-untranslated region (3’-UTR) of messenger RNAs (mRNAs) and then inhibiting the translation or inducing the degradation of mRNAs, and play ubiquitous roles in gene expression regulation contributing to diverse cellular processes such as cellular development, differentiation and apoptosis, etc.It was predicted bioinformatically that there exist more than 1000 miRNAs in human genome. The tempo-spatiality specific and tissue-specific expression patterns of miRNAs in different tissues/cells in various physiopathological conditions were well documented, and deregulated miRNAs expressions were consistently observed to involve in the initiations and developments of many diseases including human cancer. More recently, miRNAs in plasma/serum are emerging as a new class of potential biomarkers for minimally invasive diagnosis and monitoring of patients with several diseases such as cancer and cardiovascular disease. For instance, it was found that there are some abberrantly up-regulated miRNAs, such as miR-92 and miR-1, in the plasma and/or serum samples of patients with cancers or cardiovascular diseases, and therefore circulating miRNAs may serve as potential biomarkers for the diagnosis and prognosis of corresponding clinical conditions. Considering recent progresses in circulating miRNAs research field, we could speculate that the species and levels of miRNAs in plasma and/or serum may be maintained in homeostatic status under normal physiological conditions, however, if there are functional and/or metabolic abnormality or organ injuries in human body, the homeostasis of circulating miRNAs may be altered directly or indirectly and therefore result in various degree of variations of specific circulating miRNAs levels in blood.Currently, liver-related diseases such as hepatitis B virus (HBV) infection, chronic hepatitis B (CHB), liver fibrosis (LF), liver cirrhosis (LC) and hepatocellular carcinoma (HCC), etc, are still significant threats to human health worldwide. Therefore, it is still necessary to develop more specific and sensitive biomarkers to complement the traditional markers, such as ALT, AST and AFP, etc, to improve the diagnosis, assessment and health management of patients with liver pathologies.To investigate the feasibility of explorating circulating miRNAs as potential markers for the diagnosis of liver-related diseases, the present work firstly established the platform for quantifing circulating miRNAs in clinical serum samples by using real-time quantitative PCR (rt-qPCR). Thereafter, the quantitative PCR-based TaqMan low density arrays (TLDA) were employed to characterize the circulating miRNAs in the serum samples of normal control, HCC and LC patients, respectively, to investigate whether there are deregulated miRNAs in sera of patients with of liver-related diseases. Furthermore, five up-regulated miRNAs in HCC and LC serum samples, i.e. miR-146a,-215,-224,-574-3p and miR-885-5p were selected and validated in independent case-control studies. Finally, data mining based on a newly published literature was performed to understand the tissue distribution of miR-885-5p and the potential targets of miR-885-5p were predicted and analyzed by using bioinformatics softwares such as TargetScan, etc. This work achieves four main results. Firstly, Circulating miRNAs quantification platform is established in our Lab successfully. Second, The feasibility of using TLDA to quantify circulating miRNAs is evaluated. Third, Circulating miR-885-5p is found to be significantly increased in the serum samples of patients with HCC, CHB and LC, respectively. More importantly, miR-885-5p yield an AUC (area under the curve) of 0.904 in discriminating patients with liver pathologies from normal control, with specificity of 79.17% and sensitivity of 90.53%, respectively. Finally, data mining based on a newly published literature reveals that miR-885-5p is a relatively liver tissue-restricted miRNAs. Furthermore, bioinformatics analysis indicates that the ABCA1 (ATP-binding cassette sub-family A member 1, ABCA1), functioning as the gatekeeper of cholesterol reverse transport (CRT) and key modulator in phosphatide metabolism, is one of the potential targets of miR-885-5p.In summary, the technical platform established here can serve as reference for similar research related to miRNAs quantification, and more importantly, circulating miR-885-5p in serum may serve as a potential biomarker for liver-related diseases detection.更多还原