【作者】 毛启新；

【导师】 张瑾；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 背景：MircoRNAs(miRNAs)是一类短序列、非编码、具有调控功能的单链小分子RNA,长约20-24 nt,由一段具有发夹样环状结构、长约70-80 nt的单链RNA前体(pre-miRNA)经过Dicer酶加工后生成。miRNAs通过与其靶mRNA分子的3’端非编码区(3’-UTR)互补结合,在翻译水平上特异性抑制基因表达,参与调控生物生长和发育等许多复杂的生命过程。许多miRNAs的序列在起源甚远的物种之间相对保守,虽然这些miRNAs的生物学功能目前还不十分清楚,但可以肯定的是它们有不同的表达模式,同时参与调节各种生理病理过程。异常的miRNAs表达与人类某些疾病包括肿瘤的发生发展都密切相关,miRNAs被认为是一组新的癌或抑癌基因,其表达具有特异性的肿瘤组织表达谱。循环核酸(circulating nucleic acid,CNA)是指存在于血液(血清或血浆)等体液中的细胞外游离DNA和RNA,与生理和病理状态下的细胞代谢密切相关,循环DNA和RNA水平的检测及其在基因诊断等方面的应用对于恶性肿瘤等疾病的诊断和监测具有十分重要的意义。近期人们在进行循环DNA、RNA分子检测的同时,在miRNAs方面也做了许多的工作,研究结果使得与肿瘤相关的核酸分子检测这一领域变的更加让人振奋。在血液中检出肿瘤特异性的miRNAs并将其作为肿瘤的生物学标志物对于早期诊断肿瘤具有至关重要的意义。目的：1.建立起从血清中提取miRNA的方法,并检测mir-21、mir-200c、mir-205在乳腺癌患者和健康对照人群血清中的表达差异情况。2.了解mir-205对乳腺癌细胞系MCF-7生长和凋亡的影响。3.结合生物信息学技术预测miR-205的靶基因,为理解mir-205在乳腺癌发生发展中的作用提供依据。4.通过本研究,希望为乳腺癌的治疗提供新的分子靶点,为乳腺癌相关的诊断、预后标志物研究提供新的内容。方法：1.利用实时荧光定量RT-PCR技术,检测并比较mir-21、mir-200c、mir-205在乳腺癌病人和健康对照人群血清中的表达差异。2.利用脂质体转染技术,上调mir-205在MCF-7细胞中的表达。利用荧光显微镜和荧光定量RT-PCR技术检测该方法的有效性。利用MTT法分析转染前后细胞增殖活性的变化；流式细胞PI法分析转染前后细胞生长周期的变化；流式细胞仪FITC-AnnexinV/PI法分析转染前后细胞凋亡的变化。3.应用不同的生物信息学方法对在乳腺癌细胞系MCF-7中表达下调的miR-205的靶基因进行预测。结果：1.利用实时荧光定量RT-PCR法,对10例乳腺癌患者和3例健康对照人群的血清miRNA进行了检测,成功从乳腺癌患者和健康对照人群的血清中提取出miRNA,发现相对于对照组,乳腺癌患者血清中mir-200c的表达上调,mir-205表达下调,差异有统计学意义。2.本实验利用脂质体转染miRNA mimics的实验技术,其上调miRNA的有效性得到证实。发现上调mir-205表达水平以后会降低乳腺癌细胞MCF-7的增殖活性,增加细胞凋亡率,而对细胞周期无明显影响。3.应用miRBase、Pictar和Targetscan三大经典靶基因预测软件对miR-205进行靶基因预测,在至少两个以上软件预测出的靶基因有113个。其多个基因涉及细胞周期、信号转导、细胞增殖、分化和细胞凋亡等众多生物学过程,说明miR-205在乳腺癌发生发展中有着重要的生物学功能。结论：1.利用Trizol法和实时荧光RT-PCR方法能够成功提取乳腺癌患者和健康对照人群血清中的miRNA,并且发现乳腺癌患者血清中mir-200c的表达上调,mir-205表达下调,有可能作为乳腺癌早期诊断的肿瘤分子标志物。2.mir-205可降低乳腺癌细胞的增殖活性,增加凋亡率,但不影响细胞周期。3.生物信息学预测miR-205可能通过调控下游一系列靶基因的转录和翻译引起乳腺癌细胞生物学特性的改变,参与了乳腺癌的发病机制。更多还原

【Abstract】 Background:MicroRNAs(miRNAs)are a new class of non-protein-coding, endogenous, small RNAs and of about 20-24 nucleotides in length, which are cleaved from the 70-80 nt partially duplexed precursor by the RNase III Dicer. Mature miRNAs are known to negatively regulate gene expression or destroy the stability of genes via incomplete or complete matching with the 3’UTR of their target genes at the post-transcriptional level. They are involved in processes such as the regulation of organismal development, nerve differentiation, cell proliferation, apoptosis and fat metabolism. Many miRNAs have relatively conservative sequences among species. Though the functions of these miRNAs are not clear, it is sure that they have different express patterns, which can regulate many physiological and pathological processes and abnormal expression of miRNAs may result in some human diseases including tumors. miRNAs are recognized as oncogenes or anti-oncogenes and they have specific express profiles in different tumor tissues. It has been demonstrated that circulating nucleic acid (CNA) which exists in body fluids including serum and blood plasma is closely related to cell metabolism in physiological and pathological condition. The detection and gene diagnosis of CNA has played a very important role in the diagnosis of malignant tumors. Recently circulating miRNAs are found in serum and blood plasma. Thus, it is possible that circulating miRNAs become a new class of clinical biomarkers in the early detection of malignant tumors.Objective:1. To demonstrate that the extraction of RNA and subsequent identification of miRNA from serum is feasible and to detect the expression of mir-21, mir-200c and mir-205 in serum between breast cancer patients and healthy controls.2. To investigate the effect of mir-205 on cell growth and apoptosis in breast cancer cell lines.3. To predict the target genes of mir-205 using bioinformatics methods and provide theoretical support and experimental basis for further research on mechanism of mir-205 in breast cancer development. 4. In summary, this study hopes to find new molecular targets in treatment of breast cancer and new biomarkers for diagnosis and prognosis.Methods:1.Real-time quantitative PCR is used to detect the abnormal expression of mir-21, mir-200c and mir-205 in the serum of breast cancer patients compared with healthy controls.2.mir-205 mimics is transfected into MCF-7 cells and fluorescent dyes cy3 linked at the oligonucleotide was observed by fluorescence microscope and qRT-PCR of mir-205 was chosen to observe the changes of miRNA expression. MTT was tested to evaluate the changes in cell growth. Cell cycle was observed by assessment of flow cytometry PI staining and apoptosis was observed by assessment of FITC-AnnexinV/PI two-color flow cytometry before and after transfection.3.Different bioinformatic ways are used to predict the target genes of miR-205 which is down-regulated in breast cancer cell lines MCF-7.Results:1.mir-21, mir-200c and mir-205 were detected in serum of 10 breast cancer patients and 3 healthy controls with real-time quantitative PCR. Results show that mir-200c is up-regulated while mir-205 is down-regulated in breast cancer patients.2.Observation of fluorescent dyes cy3 in cells and qRT-PCR of mir-205 after transfection confirmed the efficiency of transfection. Up-regulation of mir-205 would decrease the cell activity while increasing apoptosis but has no effect on cell cycles.3.There are 113 target genes predicted by at least two bioinformatic ways, most of the genes are involved in cell cycles, cell proliferation, differentiation, apoptosis, signal transduction and so on.Conclusions:1. miRNA could be detected in serum and some miRNA abnormally expressed in serum of breast cancer patients compared with healthy controls. Circulating miRNAs may become a novel class of biomarkers for early diagnosis of breast cancer.2. mir-205 could decrease cell proliferation activity while increasing apoptosis, but has no effect on cell cycles. 3. Bioinformatic analysis that predicts miR-205 might be involved in origin and progression of breast cancer as target gene regulators.更多还原