【摘要】： 最近幾年,miRNA已成為生命科學(xué)領(lǐng)域研究的熱點,它的發(fā)現(xiàn)不僅為生物學(xué)開辟了新的領(lǐng)域,更為其生物學(xué)原理應(yīng)用于疾病治療的轉(zhuǎn)譯研究提供了新的武器,曾一度被認(rèn)為是“垃圾”RNA的小分子近十年先后7次被美國《SCIENCE》評選為“年度全球十大科技突破”。到目前為止,人們已經(jīng)發(fā)現(xiàn)了三千余種miRNAs,被認(rèn)為是最大的一類基因表達(dá)調(diào)控因子,它通過與靶基因的結(jié)合調(diào)節(jié)細(xì)胞分裂、增殖、分化和多種生物學(xué)信號通路,也可以起到腫瘤抑制基因和癌基因的作用,以及應(yīng)用在疾病診斷和基因功能研究中。 多項研究結(jié)果表明miR-122是肝臟特異的miRNA,調(diào)節(jié)脂肪酸、膽固醇的代謝和肝臟的分化,上調(diào)嗜肝病毒HCV的復(fù)制。為了研究miR-122與HBV的復(fù)制關(guān)系,我們利用生物信息學(xué)預(yù)測在HBV上1689nt-1711nt區(qū)域存在miR-122的可能靶序列。首先利用miRNA的生物學(xué)原理,將miR-122的編碼基因hcr構(gòu)建在包含綠色熒光蛋白的載體上,將此重組質(zhì)粒轉(zhuǎn)染到細(xì)胞內(nèi),hcr基因在細(xì)胞內(nèi)表達(dá),繼而在宿主細(xì)胞內(nèi)發(fā)展為成熟的miR-122,實驗中利用RT-PCR、T-A克隆技術(shù)對于細(xì)胞內(nèi)的miR-122的表達(dá)進行檢測,證實獲得能穩(wěn)定表達(dá)miR-122的真核表達(dá)細(xì)胞系。為了驗證miR-122作用HBV的靶點,首先,從HepG2215基因組DNA擴增miR-15靶序列—BCL2,插入熒光素酶報告基因(luciferase)下游的Xba1酶切位點,構(gòu)建了陽性的miR-15熒光素酶報告基因系統(tǒng),用于驗證miR-122靶點的陽性對照系統(tǒng)。已有研究證明HBV在1689nt-1711nt位置有四種轉(zhuǎn)錄本,根據(jù)miRNA的生物學(xué)原理推測miR-122可能對這四種mRNA都有作用,本實驗選用其中之一的HBx來驗證1689nt-1711nt序列與miR-122之間是否為靶向的關(guān)系。本研究構(gòu)建了包含HBx序列的熒光素酶報告基因系統(tǒng)及HBx蛋白的表達(dá)系統(tǒng),與miR-122共轉(zhuǎn)染到自身低表達(dá)miR-122的細(xì)胞系中,在過表達(dá)mirR-122、鎖定miR-122和突變miR-122三個層面上,用熒光素酶報告基因的檢測及western blot實驗方法,證實miR-122通過作用靶序列HBx對HBV復(fù)制進行負(fù)向調(diào)控,并在翻譯水平抑制HBX蛋白的表達(dá)。然后將表達(dá)HBV1.1倍體的質(zhì)粒和miR-122共轉(zhuǎn)染細(xì)胞,提取細(xì)胞內(nèi)的乙肝病毒復(fù)制中間體DNA,做southern blot檢測,結(jié)果發(fā)現(xiàn)與miR-122共轉(zhuǎn)染之后HBV復(fù)制中間體DNA的含量明顯減少,確定miR-122與HBV的復(fù)制的降低有關(guān)。
[Abstract]:In recent years, miRNA has become a hot spot in the field of life sciences. Its discovery has not only opened up a new field for biology, but also provided a new weapon for the translation of its biological principles in the study of disease therapy. Small molecules once known as "garbage" RNA have been named "Top Ten technological breakthroughs of the year" by the United States'< SCIENCE > for seven times in the past decade. So far, more than 3, 000 species of miRNAs, have been found to be the largest class of gene expression regulators, which regulate cell division, proliferation, differentiation and a variety of biological signaling pathways by binding to target genes. It can also play the role of tumor suppressor gene and oncogene, and be used in disease diagnosis and gene function research. Many studies have shown that miR-122 is a liver-specific miRNA, that regulates fatty acids, cholesterol metabolism and liver differentiation, and up-regulates the replication of hepatoviral HCV. In order to study the replication relationship between miR-122 and HBV, we used bioinformatics to predict the possible target sequences of miR-122 in the 1689nt-1711nt region on HBV. Firstly, based on the biological principle of miRNA, the encoding gene hcr of miR-122 was constructed on the vector containing green fluorescent protein. The recombinant plasmid was transfected into the cells and the hcr gene was expressed in the cells. Then the expression of miR-122 in host cells was detected by RT-PCR,T-A cloning in the mature miR-122, experiment, and the eukaryotic expression cell lines which could stably express miR-122 were obtained. In order to verify the target of miR-122 acting on HBV, firstly, the miR-15 target sequence-BCL2, was amplified from HepG2215 genome DNA and inserted into the downstream Xba1 cleavage site of luciferase reporter gene (luciferase), and a positive miR-15 luciferase reporter gene system was constructed. A positive control system used to validate miR-122 targets. Studies have shown that HBV has four transcripts at the 1689nt-1711nt site, and it is speculated that miR-122 may play a role in all four mRNA based on the biological principles of miRNA. In this study, one of the HBx was selected to verify the relationship between 1689nt-1711nt sequence and miR-122. In this study, the luciferase reporter gene system containing HBx sequence and the expression system of HBx protein were constructed. The system co-transfected with miR-122 into a cell line with low expression of miR-122 and overexpressed mirR-122,. By using luciferase reporter gene detection and western blot assay, it was confirmed that miR-122 negatively regulated HBV replication by acting on target sequence HBx, and inhibited the expression of HBX protein at the translation level on the three levels of locking miR-122 and mutated miR-122, and the expression of HBX protein was inhibited at the translation level by luciferase reporter gene detection and western blot assay. Then the plasmid expressing HBV1.1 was co-transfected with miR-122 to extract the DNA, of HBV replication intermediates in the cells for southern blot detection. The results showed that the content of DNA, the intermediate of HBV replication, was significantly decreased after co-transfection with miR-122. Determine that miR-122 is associated with reduced replication of HBV.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346

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