目录
摘要I
关键词I
AbstractII
引言
引言1
1材料与方法2
1.1 材料与仪器 2
1.1.1 研究材料 2
1.1.2 主要仪器 2
1.1.3 主要试剂 3
1.2 研究方法 3
1.2.1细胞培养3
1.2.2 双荧光素酶报告基因试验3
1.2.3细胞转染4
1.2.4 RNA的提取及逆转录4
1.2.5 qRTPCR4
1.2.6 Western Blot法对相关靶基因蛋白质水平检测5
1.2.7 内源基因表达6
2 结果与分析6
2.1 miR122靶基因预测6
2.2双荧光素酶检测试验7
2.3 Western Blot验证8
2.4 内源基因表达9
3讨论10
致谢11
参考文献 12
miR122靶基因的鉴定
摘 要
microRNA(miRNA)是一类高度保守的，与真核基因表达密切相关的、长度仅有约22个核苷酸的非编码RNA。靶标mRNA 3’端的非编码区序列(untranslated region, UTR）可与miRNA 5’端的种子区域互补配对，达到调控基因表达水平的目的。miR122是miRNA家族中重要的一员，是与肝脏相关的疾病密不可分的miRNA。本研究使用靶基因预测软件对靶基因的预测结果，选择EB1和CS两个基因进行验证，构建出包含萤火虫荧光素酶的报告基因质粒，将构建的质粒转染至HEK293T细胞和HepG2细胞中，使用双荧光素酶报告基因检测法进行验证，结果表明存在miR122时，荧光素酶活性受到显著抑制。接下来运用免疫印迹法在蛋白质水平对靶基因的准确性进行验证，将miR122 mimics转染至HEK293T细胞和HepG2细胞中，发现CS和EB1两个基因表达的蛋白质水平显著降低。同时结合了内源基因表达检测，发现靶基因与miR122的表达呈负相关关系，进一步证明EB1和CS为miR122的靶基因。本研究可 *51今日免费论文网|www.jxszl.com +Q: @351916072@ 
为下一步研究EB1和CS与miR122相关联的具体生物学功能奠定了坚实的基础。
IDENTIFICATION OF MIR122
ABSTRACT
MicroRNA (miRNA) is a type of highly conserved noncoding RNA with only 22 nucleotides, which is closely related to eukaryotic gene expression. The seed region at the 5’ end of miRNA and the untranslated region (UTR) at the 3’ end of target mRNA can recognize and complement each other to regulate gene expression and a series of physiological and pathological processes. miR122 is an important member of the miRNA family. Its specific expression in liver tissue is closely related to liver related diseases. In this study, we used target gene prediction software to predict and screen miR122 target gene, and two genes, EB1 and CS, were selected for target gene verification. A reporter gene plasmid containing firefly luciferase was constructed, and the constructed plasmid was transformed. Infected into HEK293T cells and HepG2 cells, according to the instructions using dual luciferase reporter gene detection method to verify the gene under test, the results showed that in the presence of miR122, luciferase activity was significantly inhibited. Then western blot was used to verify the accuracy of the target gene at the protein level. MiR122 mimics was transfected into HEK293T cells and HepG2 cells. It was found that the protein levels of CS and EB1 genes were significantly reduced, which was consistent with the predicted results. At the same time, combined with the detection of endogenous gene expression, it was found that there was a negative correlation between the target gene and miR122 expression, which further proved that EB1 and CS were the target genes of miR122. It can lay a solid foundation for the further study of specific biological functions of EB1 and CS associated with miR122.

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