三唑磷作用下斑马鱼miR-203和miR-217对nup43调控作用的研究

发布时间：2018-03-03 13:41

  本文选题：miR-203　切入点：miR-217　出处：《浙江理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：miRNA的作用是通过同靶mRNA的3’端靶向结合降解或者是抑制其靶基因翻译,进而可以对靶基因的转录后进行调控。另mi RNA可识别相应的靶基因且不是单一映射关系,同时存有多个不同miRNA调控同一靶基因,而miRNA也可以调控多个不同的靶基因。我们前期预测发现斑马鱼中nup43是miR-203的靶基因,并且在三唑磷,化学名称O,O-二乙基-O-(1-苯基-l,2,4-三唑-3-基)硫代磷酸酯的胁迫下nup43的表达受miR-203的调控。在本实验中,课题组利用Microcosm Targets软件预测得出nup43为miR-203与miR-217共同调控靶基因,通过实验验证了这2个miRNAs分别与nup43之间的关系。我们评估了三唑磷暴露下斑马鱼中这2个mi RNAs的表达,研究2种miRNAs能否调控nup43的表达,结果发现随着三唑磷处理浓度的升高,斑马鱼中miR-203基因的表达量显著上调,斑马鱼中miR-217基因的表达量显著下调。mi RNA可作为环境化学物质或致癌基因得生物标记物。为了解释三唑磷处理后miRNA表达的变化,进一步验证生物信息学预测的结果。我们通过将nup43 3’-UTR中miR-203和miR-217结合位点分别敲除,成功构建了2个重组海肾荧光素酶载体,分别命名为pRL/S-K.O-203、pRL/S-K.O-217。通过双荧光素酶报告基因法验证了斑马鱼nup43 3’-UTR中存在miR-217的结合位点。我们通过对ZF4细胞转染miR-217 mimic后,检测细胞中nup43的表达量,发现nup43在mRNA水平及蛋白水平都显著下调,而转染miR-217 inhibitor后,nup43的mRNA水平及蛋白水平则显著上调,但对ZF4细胞转染miR-203 mimic后,其nup43表达量为上调,但转染miR-203 inhibitor后,其nup43表达量为显著下调。依上述可得,nup43是miR-203和miR-217的靶基因,在三唑磷暴露下miR-203与miR-217的变化表明其可以作为暴露于三唑磷环境的生物标志物。
[Abstract]:The role of miRNA is to degrade or inhibit the translation of target gene by targeting the 3 '-terminal binding of target mRNA, and then to regulate the target gene after transcription. MiRNA can recognize the corresponding target gene and is not a single mapping relationship. At the same time, there are many different miRNA to regulate the same target gene, and miRNA can also regulate many different target genes. We predicted that nup43 in zebrafish is the target gene of miR-203, and in triazophos, The expression of nup43 was regulated by miR-203 under the stress of OO2-diethyl-O-1-phenyl-1-phenyl-1-phenyl-1-phenyl-1-phenyl-1-diethyl-1-triazolyl) phosphate. In this experiment, we predicted that nup43 is a target gene regulated by miR-203 and miR-217 by Microcosm Targets software. We evaluated the expression of miRNAs in zebrafish exposed to triazophos and studied whether the two miRNAs could regulate the expression of nup43. The results showed that the expression of nup43 increased with the concentration of triazophos. The expression of miR-203 gene in zebrafish was upregulated, and the expression of miR-217 gene in zebrafish was down-regulated. Mi RNA could be used as an environmental chemical or a biomarker of oncogene, in order to explain the change of miRNA expression after triazophos treatment. The results of bioinformatics prediction were further verified. By knockout the binding sites of miR-203 and miR-217 in nup43 3, we successfully constructed two recombinant marine kidney luciferase vectors. They were named as pRL / S-K.O-203 and pRL / S-K.O-217.The binding sites of miR-217 in zebrafish nup43 3O -UTR were verified by double luciferase reporter gene method. After transfection of ZF4 cells into miR-217 mimic, we detected the expression of nup43 in ZF4 cells. It was found that nup43 was significantly down-regulated in mRNA and protein levels, while mRNA and protein levels of nup43 were up-regulated after transfection of miR-217 inhibitor, but nup43 expression was up-regulated in ZF4 cells after transfection of miR-203 mimic, but after transfection of miR-203 inhibitor. Nup43 was the target gene of miR-203 and miR-217. The changes of miR-203 and miR-217 under triazophos exposure indicated that it could be used as a biomarker of triazophos exposure.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X174;Q78

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