法洛氏四联症患者心脏miRNA表达分析及通用TaqMan探针检测miRNA方法的建立和应用

【作者】 罗欣；

【导师】 马端；

【作者基本信息】 复旦大学 ， 分子医学， 2011， 硕士

【摘要】 第一部分微小RNA (micro-RNA, miRNA)是一类长约22-25nt的非编码功能小RNA,它在许多生物中都有表达。miRNA原初转录本(pri-miRNA)经过核内和胞质中一系列的加工修饰后形成成熟的miRNA。首先,miRNA基因在RNA polymeraseⅡ作用下,转录生成pri-miRNA;然后在内切酶Drosha作用下,pri-miRNA释放出60-70nt的前体(pre-miRNA),后者具有不完美的发卡结构；最后,pre-miRNA在xportin-5作用下进入胞质,并在Dicer作用下,形成成熟miRNA。成熟的miRNA通过与靶基因3’非翻译区(3’-UTR区)形成不完全配对,从而抑制靶基因翻译,或形成接近完全的配对,导致靶(?)nRNA降解。miRNA基因的数目大约为蛋白编码基因数目的1%,但是它可以调控大约30%的人类基因,这说明miRNA是通过复杂的调控网络发挥功能。miRNA在很多生物的生理和病理过程中发挥重要作用,影响细胞发育、增殖、调亡,并与许多疾病相关,如先天性心脏病、肿瘤等。越来越多的证据表明,miRNA在心脏发育中起重要的作用,并与许多先天性心脏病的发病机制相关。miRNA是目前研究的一个热点,对于它的研究无论是在阐明疾病的发生机制还是在将来的临床应用中都有重要意义。法洛氏四联症(tetralogy of Fallot, TOF)又称发绀四联症,是一种联合的先天性心脏血管畸形,包括室间隔缺损、肺动脉狭窄、主动脉骑跨和右心室肥厚。本病是最常见的紫绀型先天性心脏血管病,是由环境因素和遗传因素相互作用引起的复杂性遗传病,发病率约占先天性心脏病的10%-15%,男女比例相仿。目前已发现多个与法洛氏四联症发病相关的致病基因。但是因为法洛氏四联症是由多基因导致的复杂性先天性疾病,目前并不能确定其确切的致病基因。迄今为止对于法洛氏四联症的发病机制并没有有关miRNA(?)的研究。研究目的寻找法洛氏四联症相关miRNA,确定其靶基因。方法和结果(1)应用博奥公司Affymetrix GeneChip miRNA Array芯片在3例正常对照样本和5病例法洛氏四联症患者心脏样本进行miRNA筛选。根据软件运算和人工比对,我们共选定了40个候选miRNA(?)进行下一步的验证工作。(2)我们在6例正常对照样本和26例法洛氏四联症患者心脏样本中用实时定量PCR方法验证芯片筛选结果,发现13个miRNA(?)对照组和病例组中存在表达差异,p<0.05。(3)因为样本收集的困难,对照组和病例组样本间存在年龄差异,而miRNA的可以根据发育阶段的不同存在表达差异。为了排除年龄差异造成的影响,我们在10天龄C57小鼠和2-3个月C57小鼠的心脏组织中对结果进行检测,发现8个miRNA存在随年龄变化表达量存在差异。(4)为了寻找(?)niRNA对法洛氏四联症发病的发病机制的影响,我们预测了表达量变化的miRNA的可能的靶基因,并通过双荧光报告基因方法对靶基因进行了初步筛选。结论在法洛氏四联症的患者中,miR-363、miR-424、miR-424*、miR-181c和miR-181d的表达上调。并验证NF1和HAS2是miR-424的靶基因；PAX3和HAS2是miR-363的靶基因。第二部分近年来miRNA对靶基因的调节作用的研究已成为热点,大多数miRNA功能还不清楚,所以建立敏感特异的检测方法,并了解各种：niRNA在组织中的时空分析情况,是理解其作用的关键步骤。传统的Northern Blot等方法由于操作的复杂性和低敏感性逐渐被高通量的,灵敏的检测方法-实时定量PCR-所代替。但是由于miRNA长度只有22-25nt,很难用常规方法检测,只有通过特殊的设计,所以miRNA检测方法成为其功能研究中的一大难题。目前用于检测miRNA的RT-PCR方法主要有：茎环PCR方法、延长引物PCR方法和通用引物方法。其中前两种都是根据特定的miRNA序列设计反转录引物,还需要根据特定的反转录引物进行miRNA的检测,不仅工作量大,而且费用高昂。现在常用的为通用引物RT-PCR方法,我们在此基础上进行了改进,使通用反转录引物序列加长并且本身末端可以形成茎环结构,目的是防止在低退火温度时引物本身互补,而且可以根据此序列设计通用下游引物和通用检测探针序列。在实时定量PCR检测方法中,TaqMan探针法和SYBR Green法都是比较常用的方法。TaqMan探针具有高度的特异性和准确性,但是由于要针对每个检测目标设计探针,其价价格昂贵,从而限制了它的的应用。而传统SYBR Green染料法的特异型低,荧光染料能和任何dsDNA结合。因此它也能与非特异的dsDNA(如引物二聚体)结合,使其实验容易产生假阳性信号,但是由于操作方便和成本较低而受到广泛的青睐。所以,对于TaqMan探针法和SYBR Green法的比较,并不能确定哪种方法占有绝对的优势。为了寻找一种既可以保证检测的特异性,又可可以减低成本的方法,我们设计了一条通用的TaqMan探针,可以结合特异的上游引物和通用引物对miRNA进行检测。研究目的建立通用TaqMan探针检测miRNA方法。方法和结果(1)根据通用的反转录引物设计TaqMan探针序列,并保证此探针序列位于通用下游引物之前。(2)通过对已报道的miRNA组织特异性miRNA的检测,证实新的通用TaqMan探针的可行性,并且有更大的精确检测范围。(3)通过对心脏组织特异性(?)niR-1-1和niR-133a的检测,证实新的TaqMan探针可以区分组织特异性表达的miRNA。(4)通过与SYBR Green方法的比较,结果证明新的TaqMan方法可以避免假阳性信号的影响。(5)我们用通用TaqMan探针方法在5种组织中检测了39种miRNA的表达,证实此方法可以用于miRNA的中等量检测。结论我们设计了一种新的依赖于通用TaqMan探针的miRNA检测方法。此方法不仅保留了传统TaqMan探针检测的特异性,避免了SYBR Green法可能造成的假阳性结,有更大的检测范围,而且可以在组织特异性miRNA检测中应用。更多还原

【Abstract】 Part OneMicroRNAs (miRNAs) are endogenous 22-25 nucleotides that are processed from larger hairpin precursors, whose function is as regulator of gene expression. Pri-miRNAs which often contain 7mGpppG and polyA are transcript by RNA polymerasesⅡin nucleolus. Then the stem loop intermediate pre-miRNAs have been liberated performed by Drosha RNaselll endonucleas. These pre-miRNAs can be transported to cytoplasm by RAN-GTP and exportin-5, where mature miRNA is cleavage by Dicer from one arm of them. Mature miRNA combines with the complementary base of mRNA to regulate gene expression through inhibiting translation or degrading specific mRNA.MiRNAs are believed to participate in variedly regulation approaches, including cell development, ulation of hematopoietic lineage differentiation, cardiovascular biology and human tumorgenesis.Tetralogy of Fallot (TOF) is the most common heart defect in children which occurs at approximately 7%-8% of congenital heart defects (CHD). Infants with this abnormality develop signs of the condition very early in life, including Pulmonary stenosis, Overriding aorta, ventricular septal defect (VSD) and Right ventricular hypertrophy. Although their etiology is often poorly understood, most are considered to arise from multifactorial influences, including environmental and genetic components. MiRNAs have been paid great attention in resent research. But to our best of knowledge, microRNA expression on TOF in human has not been previously reported.ObjectionFind out the TOF-associated miRNA and determine the target gene.Methods and results(1) MiRNAs microarray was used to detect miRNA expression of 3 normal and 5 TOF heart tissues. Among the screened microRNAs,40 signals have different experssion between normal persons and TOF children.(2) Expression of all the selected microRNAs by q-RT-PCR from more TOF patients (n=26) and normal persons (n=6). The analysis confirmed that 13 microRNAs have differently expression patterns and contain 10 up-regulated and 3 down-regulated. (3) Since the control samples’ages were older than the TOF samples, there may have differences in miRNAs expression with age changes. To avoid this false positive result,2-3month and 10 days C57 mouse heart tissues was used to correct the outcome acquired by q-RT-PCR. There are 8 miRNAs which growth and development have effect on.(4) To analysis the biological significance of miRNA deregulation, we use bioinformatics tools for target gene prediction of miRNAs verified by q-RT-PCR from the Candidate gene library of congenital heart disease and verified in vitro by luciferase array.Conclusions:The miR-363, miR-424, miR-424*, miR-181c and miR-181d were up-regulated in Tetralogy of Fallot and luciferase activity of NF1 and HAS2 3’-UTR was inhibited 50% and 40% by mir-424, while PAX3 and HAS2 3-UTR was inhibited 40% by mir-363.Part TwoThe gene regulation mechanisms of miRNAs have had great advancement since the discovery of these small non-encoding molecules. Therefore, methods for identification and quantified detection of miRNAs are important in research.The conditional miRNA detection contains two:1) Northern blot:this method often needed plentiful RNA and isotope.2) Quantitative real-time PCR (q-RT PCR) assays:this method has been proven to be a sensitive and specific tool for miRNA expression, which contains probe-based (TaqMan probe array) and probe-less (SYBR Green array) methods. SYBR Green is a dye that binds to all double-stranded DNA molecules, and it may overestimate the quantity of target because the formation of primer dimer can give the false results. In contrast, the TaqMan probe may target the DNA with a particular region based on its probe hydrolysis. Even it had high specificity for miRNA detection, but it is expressive to design each target probe in abundant experimental use. Since either of them has shortcomings, new method to detect miRNAs are needed for the research.Objection:Send up a universal TaqMan probe method to detect miRNAs.Methods and results (1) The TaqMan probe was designed and according to the modified anchor primer and in front of the localization of the universal reverse primer.(2) To examine the feasibility of the novel, specific TaqMan probe, miR-126 was used as a positive control to detect.(3) MiR-1-1 and miR-133a was used to determine the miRNA expression level in tissue by the modified TaqMan probe.(4) Compared with SYBR Green method, it is not affect by the primer dimer as the traditional TaqMan probe array and would precise quantification depend on the template concentration with a larger region.(5) The modified TaqMan probe was used to discover miRNA expression profile in five different tissues.ConclusionsWe present a modified TaqMan probe q-RT PCR method for easy and accurate monitoring of miRNAs. This method bases on the modified polyA RT-PCR and use universal TaqMan probe to detect miRNAs.更多还原