细胞质、细胞核、线粒体及细胞外microRNAs的功能及应用研究

发布时间：2018-05-18 06:59

  本文选题：晚期糖基化终产物 + miR-214　； 参考：《南京大学》2012年博士论文

【摘要】：MicroRNA(miRNA)是一类内源性非编码的小分子单链RNA,长约19-23碱基。MiRNA通过与目标mRNA分子的3'端非编码区域(3'-UTR)结合抑制靶基因的翻译或导致靶基因mRNA的降解,从而在转录后水平调控基因的表达。大量的研究表明,miRNAs不但参与生物体多种基本生命过程的调控,它还与疾病的发生发展存在千丝万缕的联系,在生命活动中起着非常重要的作用。但是,与新的miRNA的频频发现相比,miRNA功能及应用方面的研究却相对缓慢。本论文主要就以下六个方面对miRNA功能进行研究:第一方面是成熟miRNA功能的鉴定及其对疾病的影响;第二方面是miRNA在细胞核的存在及其作用方式和功能;第三方面是miRNAs在线粒体的存在及其可能的生物学功能;第四方面是血清/血浆miRNA作为非侵入地诊断诊断标志物方面的研究;第五方面是血清/血浆miRNA稳定的机制,第六方面是miRNAs对免疫抑制细胞的影响。主要研究成果如下:1.miRNA-214通过抑制PTEN的表达而延迟单核细胞凋亡。我们研究发现了 miR-214以及它的靶基因在晚期糖基化终产物(AGE)诱导的单核细胞凋亡延迟中的作用。通过利用miRNA芯片和茎-环qRT-PCR研究了 AGEs处理/不处理的单核细胞系THP-1 miRNA表达谱,发现miR-214在AGE处理的THP-1和人外周血单核细胞中都是显著升高的,并且AGE诱导的单核细胞miR-214表达的升高很可能是通过AGEs的受体的。另外,我们发现在AGEs含量很高的慢性肾衰竭病人的单核细胞中miR-214也是显著升高的。然后,通过靶点预测和荧光素酶报告基因实验我们进一步发现miR-214能够和PTEN mRNA的3'UTR特异性的结合,表明PTEN是miR-214的一个靶基因。通过比较正常单核细胞、AGE处理的单核细胞核慢性肾衰竭病人的单核细胞发现PTEN的降低可以延迟单核细胞的凋亡。在THP-1中过表达miR-214抑制PTEN的表达也延迟THP-1细胞的凋亡,而抑制miR-214表达则基本上消除了 AGEs诱导的细胞凋亡的延迟。2.证明miR-709在细胞核中直接在转录后水平调节miR-15a/16-1的生成首次证明miR-709主要存在于多种细胞的细胞核中并且其在细胞核的分布随着凋亡的刺激而迅速发生改变。另外,miR-709在细胞核中直接和pri-miR-15a/16-1上的19-nt的高度互补序列结合从而抑制pri-miR-15a/16-1加工成为pre-miR-15a/16-1导致miR-15a/16-1成熟的减少。更重要的是,miR-709还通过miR-16-1/Bcl2通路参与细胞凋亡的调节。我们的研究是对miRNAs经典的作用方式的一个补充。3.首次发现小鼠肝脏线粒体相关的miRNAs以及它们可能的生物学功能尽管我们已经在细胞核中发现了成熟的miRNAs,但是诸如线粒体的细胞器中是否有特定的miRNAs还不是很清楚。我们通过microRNA微阵列芯片技术在基因组水平分析miRNAs表达谱并且通过茎环miRNA的qRT-PCR方式进行验证,发现了线粒体中特异并且富集的miRNAs——miR-705、miR-202-5p、miR-134,这些线粒体miRNAs可能参与调节线粒体基因的表达以及其他细胞过程,例如:细胞凋亡、增殖和分化。4.首次证明血清miRNA能够作为HBV感染和HBV阳性的肝细胞癌诊断标志物近年来,发现miRNA除了存在于细胞或组织(细胞质、线粒体、细胞核、p body)中,在人的血清、血浆等体液中也存在大量的miRNAs,并且这些miRNAs能够反映一定的生理状态。我们搜集了513个血清标本(210例正常对照、135例HBV感染、48例HCV感染、120例HCC感染),利用Solexa测序初筛和qRT-PCR验证的实验策略以期找到乙肝及相关疾病的标志miRNAs。首先,由于慢性乙肝和肝细胞癌之间的密切关系,我们比较了正常对照血清和乙肝患者血清miRNAs表达谱,得到了 13个HBV异常表达的miRNAs。这13个miRNA标志物不仅能够准确将HBV和正常对照、HCV区分开,也能将HBV阳性的HCC和正常对照及HBV区分开。其次,我们还直接比较了正常对照和肝细胞癌的miRNA表达谱,发现6个miRNAs在肝细胞癌血清样本中是显著升高的,有意思的是,这些miRNAs中的两个miRNA:miR-375和miR-92a也是HBV特异的。5.发现Ago2选择性地保护MVs中的miRNAs并且介导其功能循环miRNAs具有很高的稳定性,它能够作为多种疾病的生物标志物或者在细胞之间充当信号分子的作用。然而,循环miRNAs稳定的机制到目前为止还不是很不清楚。我们研究发现Ago2蛋白复合物能够选择性地保护MVs中的miRNAs,并且能介导与之结合的miRNAs功能。6.发现miR-155和miR-21可能介导骨髓来源的免疫抑制细胞MDSC的增殖、激活及功能最后我们又将研究回归到miRNAs最本质的问题上——通过调节蛋白表达来发挥功能,通过研究我们发现不管是小鼠骨髓或者是人的细胞系体外诱导还是肿瘤小鼠的骨髓来源的抑制细胞产生过程中miR-155和miR-21表达量都是增加的,提示这两个miRNAs在MDSC增殖、激活和发挥功能过程中是有潜在作用的。目前该研究还处于起步阶段,我还将作进一步深入的研究。综上所述,本论文首先从经典的细胞质miRNA转录后调控靶基因的作用方式入手,研究了 miR-214在细胞凋亡中的作用;其次发现细胞核特异的miR-709能够在转录后水平调节miR-15a/16-1的生成,并通过miR-16/Bcl2通路参与细胞凋亡的调控,扩大了 miRNAs的功能;进一步研究我们还发现miRNAs还存在于线粒体中并具有介导线粒体功能的潜能;同时我们发现细胞和组织外的循环系统血清中的miRNAs能够作为潜在的HBV和HBV-HCC诊断标志物;最后我们找到了主要存在于MV中的血清miRNAs稳定的机制。总之,我们通过方方面面的研究,将miRNA放在细胞质、细胞核、线粒体、细胞外的循环系统中观察规律,发现miRNA在细胞的不同层面上都与凋亡、代谢等重要的生理功能相关,并具有重大的临床应用价值。
[Abstract]:MicroRNA (miRNA) is a class of endogenous non coding small molecule single strand RNA. Long approximately 19-23 base.MiRNA can inhibit the translation of the target gene by combining with the 3'end non coding region (3'-UTR) of the target mRNA molecule, or lead to the degradation of the target gene mRNA, thus regulating the expression of the gene at the post transcriptional level. A large number of studies have shown that miRNAs not only participates in the organism. The regulation of various basic life processes is also linked with the occurrence and development of the disease. It plays a very important role in life activities. However, compared with the frequency discovery of the new miRNA, the research of miRNA function and application is relatively slow. This paper is to study the miRNA function in the following six aspects: The first is the identification of the mature miRNA function and its impact on the disease; the second is the existence of miRNA in the nucleus and its function and function; the third is the existence of miRNAs in the mitochondria and its possible biological functions; the fourth is the study of serum / plasma miRNA as a noninvasive diagnostic marker; The five is the stable mechanism of serum / plasma miRNA, and the sixth is the effect of miRNAs on immunosuppressive cells. The main research results are as follows: 1.miRNA-214 delayed the apoptosis of monocytes by inhibiting the expression of PTEN. We found that miR-214 and its target genes were induced by late glycosylation end products (AGE) to induce monocyte apoptosis By using miRNA chip and stem ring qRT-PCR, the THP-1 miRNA expression profiles of AGEs treated / non processing mononuclear cell lines were studied. It was found that miR-214 was significantly increased in THP-1 and human peripheral blood mononuclear cells treated by AGE, and the increase of AGE induced mononuclear cell miR-214 was probably through AGEs receptor. In addition, we found that miR-214 was also significantly elevated in the monocytes of patients with chronic renal failure with high AGEs content. Then, we further identified the combination of miR-214 and the 3'UTR specificity of PTEN mRNA through target prediction and luciferase reporter gene experiment, indicating that PTEN is a target gene for miR-214. Mononuclear cells, mononuclear cells of AGE treated mononuclear cell nuclear chronic renal failure patients found that the decrease of PTEN could delay the apoptosis of monocyte. Overexpression of miR-214 inhibiting PTEN expression in THP-1 also delayed the apoptosis of THP-1 cells, and the inhibition of miR-214 expression was basically eliminating the delay.2. of AGEs induced apoptosis, which proved miR-709. The production of miR-15a/16-1 directly at the post transcriptional level in the nucleus for the first time shows that miR-709 is mainly in the nucleus of a variety of cells and its distribution in the nucleus changes rapidly with the stimulation of apoptosis. In addition, miR-709 is combined in the nucleus of the nucleus directly with the highly complementary sequence of 19-nt on pri-miR-15a/ 16-1. The inhibition of pri-miR-15a/16-1 processing as a result of the decrease in the maturation of miR-15a/16-1 by pre-miR-15a/16-1. More importantly, miR-709 is also involved in the regulation of apoptosis through the miR-16-1/Bcl2 pathway. Our study is a supplement to the miRNAs classic mode of action for the first time in the discovery of mitochondrial related miRNAs in the liver of mice and their possible effects. Biological function, although we have found mature miRNAs in the nucleus, but it is not clear whether there is a specific miRNAs in the organelles of the mitochondria. We have analyzed the miRNAs expression profiles at the genomic level by microRNA microarray technology and verified by the qRT-PCR mode of the stem ring miRNA. Specific and enriched miRNAs - miR-705, miR-202-5p, miR-134, these mitochondrial miRNAs may be involved in regulating the expression of mitochondrial genes and other cellular processes, such as cell apoptosis, proliferation and differentiation.4. for the first time that serum miRNA can be used as a diagnostic marker for HBV infection and HBV positive hepatocellular carcinoma, and found miRNA in recent years. In addition to the presence in cells or tissues (cytoplasm, mitochondria, nuclei, P body), there are also a large number of miRNAs in human serum, plasma and other body fluids, and these miRNAs can reflect a certain physiological state. We have collected 513 serum specimens (210 normal controls, 135 HBV infection, 48 HCV infection, 120 HCC infection), and Solexa test. The experimental strategy of preface screening and qRT-PCR validation was expected to find the marker miRNAs. of hepatitis B and related diseases first. Due to the close relationship between chronic hepatitis B and hepatocellular carcinoma, we compared the miRNAs expression profiles of normal control sera and hepatitis B patients, and obtained the 13 miRNA markers of 13 HBV abnormal tables, which are not only accurate. It is true that HBV and normal controls, separated by HCV, can also separate HBV positive HCC from normal controls and HBV. Secondly, we have compared the miRNA expression profiles of normal controls and hepatocellular carcinoma, and found that 6 miRNAs in the serum samples of hepatocarcinoma were significantly higher, and that is, two miRNA:miR-375 and miR-92a in these miRNAs. HBV specific.5. also found that Ago2 selectively protects miRNAs in MVs and mediates its functional cycle miRNAs with high stability. It can act as a biomarker of a variety of diseases or act as a signal molecule between cells. However, the mechanism of circulating miRNAs stabilization is not so unclear so far. It was found that the Ago2 protein complex could selectively protect miRNAs in MVs and mediate the combination of miRNAs function.6. to discover that miR-155 and miR-21 may mediate the proliferation, activation, and function of bone marrow derived immunosuppressive cells MDSC, and finally we will return to the problem of the most essential of miRNAs - by regulating protein expression. We have found that the expression of miR-155 and miR-21 in the production of miR-155 and miR-21 in the bone marrow source of tumor mice is increased by studying in vitro induction of mouse bone marrow or human cell lines, suggesting that these two miRNAs are potential for the proliferation, activation and functional process of MDSC. It is still in the initial stage, and I will do further research. To sum up, first of all, this thesis begins with the classical cytoplasmic miRNA transcriptional regulation of the target gene, and studies the role of miR-214 in the apoptosis. Secondly, it is found that the nuclear specific miR-709 can regulate the generation of miR-15a/16-1 at the post transcriptional level. MiR-16/Bcl2 pathway participates in the regulation of apoptosis, enlarges the function of miRNAs; further studies have found that miRNAs still exists in mitochondria and has the potential to mediate mitochondrial function; meanwhile, we found that miRNAs in the serum of cells and tissues outside the tissues can be used as a potential diagnostic marker for HBV and HBV-HCC; After that, we found the mechanism of serum miRNAs stability mainly in MV. In a word, we put miRNA in the cytoplasm, nucleus, mitochondria, and extracellular circulatory system through various aspects, and found that miRNA is related to the important physiological functions such as withering and metabolism at different levels of the cell, and it is of great importance. The clinical application value.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R3416;Q23
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本文编号：1904882