本文選題：晚期糖基化終產(chǎn)物 + miR-214�。� 參考：《南京大學(xué)》2012年博士論文

【摘要】：MicroRNA(miRNA)是一類內(nèi)源性非編碼的小分子單鏈RNA,長(zhǎng)約19-23堿基。MiRNA通過(guò)與目標(biāo)mRNA分子的3'端非編碼區(qū)域(3'-UTR)結(jié)合抑制靶基因的翻譯或?qū)е掳谢騧RNA的降解,從而在轉(zhuǎn)錄后水平調(diào)控基因的表達(dá)。大量的研究表明,miRNAs不但參與生物體多種基本生命過(guò)程的調(diào)控,它還與疾病的發(fā)生發(fā)展存在千絲萬(wàn)縷的聯(lián)系,在生命活動(dòng)中起著非常重要的作用。但是,與新的miRNA的頻頻發(fā)現(xiàn)相比,miRNA功能及應(yīng)用方面的研究卻相對(duì)緩慢。本論文主要就以下六個(gè)方面對(duì)miRNA功能進(jìn)行研究:第一方面是成熟miRNA功能的鑒定及其對(duì)疾病的影響;第二方面是miRNA在細(xì)胞核的存在及其作用方式和功能;第三方面是miRNAs在線粒體的存在及其可能的生物學(xué)功能;第四方面是血清/血漿miRNA作為非侵入地診斷診斷標(biāo)志物方面的研究;第五方面是血清/血漿miRNA穩(wěn)定的機(jī)制,第六方面是miRNAs對(duì)免疫抑制細(xì)胞的影響。主要研究成果如下:1.miRNA-214通過(guò)抑制PTEN的表達(dá)而延遲單核細(xì)胞凋亡。我們研究發(fā)現(xiàn)了 miR-214以及它的靶基因在晚期糖基化終產(chǎn)物(AGE)誘導(dǎo)的單核細(xì)胞凋亡延遲中的作用。通過(guò)利用miRNA芯片和莖-環(huán)qRT-PCR研究了 AGEs處理/不處理的單核細(xì)胞系THP-1 miRNA表達(dá)譜,發(fā)現(xiàn)miR-214在AGE處理的THP-1和人外周血單核細(xì)胞中都是顯著升高的,并且AGE誘導(dǎo)的單核細(xì)胞miR-214表達(dá)的升高很可能是通過(guò)AGEs的受體的。另外,我們發(fā)現(xiàn)在AGEs含量很高的慢性腎衰竭病人的單核細(xì)胞中miR-214也是顯著升高的。然后,通過(guò)靶點(diǎn)預(yù)測(cè)和熒光素酶報(bào)告基因?qū)嶒?yàn)我們進(jìn)一步發(fā)現(xiàn)miR-214能夠和PTEN mRNA的3'UTR特異性的結(jié)合,表明PTEN是miR-214的一個(gè)靶基因。通過(guò)比較正常單核細(xì)胞、AGE處理的單核細(xì)胞核慢性腎衰竭病人的單核細(xì)胞發(fā)現(xiàn)PTEN的降低可以延遲單核細(xì)胞的凋亡。在THP-1中過(guò)表達(dá)miR-214抑制PTEN的表達(dá)也延遲THP-1細(xì)胞的凋亡,而抑制miR-214表達(dá)則基本上消除了 AGEs誘導(dǎo)的細(xì)胞凋亡的延遲。2.證明miR-709在細(xì)胞核中直接在轉(zhuǎn)錄后水平調(diào)節(jié)miR-15a/16-1的生成首次證明miR-709主要存在于多種細(xì)胞的細(xì)胞核中并且其在細(xì)胞核的分布隨著凋亡的刺激而迅速發(fā)生改變。另外,miR-709在細(xì)胞核中直接和pri-miR-15a/16-1上的19-nt的高度互補(bǔ)序列結(jié)合從而抑制pri-miR-15a/16-1加工成為pre-miR-15a/16-1導(dǎo)致miR-15a/16-1成熟的減少。更重要的是,miR-709還通過(guò)miR-16-1/Bcl2通路參與細(xì)胞凋亡的調(diào)節(jié)。我們的研究是對(duì)miRNAs經(jīng)典的作用方式的一個(gè)補(bǔ)充。3.首次發(fā)現(xiàn)小鼠肝臟線粒體相關(guān)的miRNAs以及它們可能的生物學(xué)功能盡管我們已經(jīng)在細(xì)胞核中發(fā)現(xiàn)了成熟的miRNAs,但是諸如線粒體的細(xì)胞器中是否有特定的miRNAs還不是很清楚。我們通過(guò)microRNA微陣列芯片技術(shù)在基因組水平分析miRNAs表達(dá)譜并且通過(guò)莖環(huán)miRNA的qRT-PCR方式進(jìn)行驗(yàn)證,發(fā)現(xiàn)了線粒體中特異并且富集的miRNAs——miR-705、miR-202-5p、miR-134,這些線粒體miRNAs可能參與調(diào)節(jié)線粒體基因的表達(dá)以及其他細(xì)胞過(guò)程,例如:細(xì)胞凋亡、增殖和分化。4.首次證明血清miRNA能夠作為HBV感染和HBV陽(yáng)性的肝細(xì)胞癌診斷標(biāo)志物近年來(lái),發(fā)現(xiàn)miRNA除了存在于細(xì)胞或組織(細(xì)胞質(zhì)、線粒體、細(xì)胞核、p body)中,在人的血清、血漿等體液中也存在大量的miRNAs,并且這些miRNAs能夠反映一定的生理狀態(tài)。我們搜集了513個(gè)血清標(biāo)本(210例正常對(duì)照、135例HBV感染、48例HCV感染、120例HCC感染),利用Solexa測(cè)序初篩和qRT-PCR驗(yàn)證的實(shí)驗(yàn)策略以期找到乙肝及相關(guān)疾病的標(biāo)志miRNAs。首先,由于慢性乙肝和肝細(xì)胞癌之間的密切關(guān)系,我們比較了正常對(duì)照血清和乙肝患者血清miRNAs表達(dá)譜,得到了 13個(gè)HBV異常表達(dá)的miRNAs。這13個(gè)miRNA標(biāo)志物不僅能夠準(zhǔn)確將HBV和正常對(duì)照、HCV區(qū)分開(kāi),也能將HBV陽(yáng)性的HCC和正常對(duì)照及HBV區(qū)分開(kāi)。其次,我們還直接比較了正常對(duì)照和肝細(xì)胞癌的miRNA表達(dá)譜,發(fā)現(xiàn)6個(gè)miRNAs在肝細(xì)胞癌血清樣本中是顯著升高的,有意思的是,這些miRNAs中的兩個(gè)miRNA:miR-375和miR-92a也是HBV特異的。5.發(fā)現(xiàn)Ago2選擇性地保護(hù)MVs中的miRNAs并且介導(dǎo)其功能循環(huán)miRNAs具有很高的穩(wěn)定性,它能夠作為多種疾病的生物標(biāo)志物或者在細(xì)胞之間充當(dāng)信號(hào)分子的作用。然而,循環(huán)miRNAs穩(wěn)定的機(jī)制到目前為止還不是很不清楚。我們研究發(fā)現(xiàn)Ago2蛋白復(fù)合物能夠選擇性地保護(hù)MVs中的miRNAs,并且能介導(dǎo)與之結(jié)合的miRNAs功能。6.發(fā)現(xiàn)miR-155和miR-21可能介導(dǎo)骨髓來(lái)源的免疫抑制細(xì)胞MDSC的增殖、激活及功能最后我們又將研究回歸到miRNAs最本質(zhì)的問(wèn)題上——通過(guò)調(diào)節(jié)蛋白表達(dá)來(lái)發(fā)揮功能,通過(guò)研究我們發(fā)現(xiàn)不管是小鼠骨髓或者是人的細(xì)胞系體外誘導(dǎo)還是腫瘤小鼠的骨髓來(lái)源的抑制細(xì)胞產(chǎn)生過(guò)程中miR-155和miR-21表達(dá)量都是增加的,提示這兩個(gè)miRNAs在MDSC增殖、激活和發(fā)揮功能過(guò)程中是有潛在作用的。目前該研究還處于起步階段,我還將作進(jìn)一步深入的研究。綜上所述,本論文首先從經(jīng)典的細(xì)胞質(zhì)miRNA轉(zhuǎn)錄后調(diào)控靶基因的作用方式入手,研究了 miR-214在細(xì)胞凋亡中的作用;其次發(fā)現(xiàn)細(xì)胞核特異的miR-709能夠在轉(zhuǎn)錄后水平調(diào)節(jié)miR-15a/16-1的生成,并通過(guò)miR-16/Bcl2通路參與細(xì)胞凋亡的調(diào)控,擴(kuò)大了 miRNAs的功能;進(jìn)一步研究我們還發(fā)現(xiàn)miRNAs還存在于線粒體中并具有介導(dǎo)線粒體功能的潛能;同時(shí)我們發(fā)現(xiàn)細(xì)胞和組織外的循環(huán)系統(tǒng)血清中的miRNAs能夠作為潛在的HBV和HBV-HCC診斷標(biāo)志物;最后我們找到了主要存在于MV中的血清miRNAs穩(wěn)定的機(jī)制�？傊�,我們通過(guò)方方面面的研究,將miRNA放在細(xì)胞質(zhì)、細(xì)胞核、線粒體、細(xì)胞外的循環(huán)系統(tǒng)中觀察規(guī)律,發(fā)現(xiàn)miRNA在細(xì)胞的不同層面上都與凋亡、代謝等重要的生理功能相關(guān),并具有重大的臨床應(yīng)用價(jià)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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R3416;Q23
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本文編號(hào)：1904882