Dlk1-Dio3印记区域内CGI-2调控基因表达的研究

发布时间：2018-04-16 11:05

  本文选题：Dlk1-Dio3印记区域 + 差异甲基化区域　； 参考：《哈尔滨工业大学》2016年硕士论文

【摘要】：Dlk1-Dio3印记基因簇位于小鼠的第十二号染色体末端(12qF1),以及人类的第14号染色体上(14q32)。该印记区域对胚胎和胎盘的生长发育起到重要的调控作用,还影响成体的新陈代谢和大脑的功能。且该区域内各基因表达在生长发育中呈现一个动态变化的过程。CGI-2位于该区域内miR-341和miR-1188附近,为该区域内首个被发现的母本甲基化而父本非甲基化的差异甲基化区域,且为结合CTCF并具有绝缘子活性的DNA功能原件。因此,本文选取Dlk1-Dio3印记区域为研究对象,采用CRISPR/Cas9基因编辑技术对CGI-2进行长片段的基因组删除,在细胞水平上分析CGI-2敲除后其上下游基因的表达变化。首先,本文对细胞系内Dlk1-Dio3印记区域中各基因表达水平进行分析,从而筛选出三株适合敲除实验的细胞系。所选择的三株细胞系分别为Dlk1-Dio3印记区域中各基因表达水平偏高的N2a、该印记区域中各基因表达水平较低的Hepa1-6以及非癌细胞系NIH3T3。通过向导RNA预测网站设计出用于CRISPR/Cas9敲除实验的两对向导RNA,并且构建px330重组质粒。其次,在N2a细胞中进行CGI-2敲除预实验,选择适合用于实验的向导RNA。随后,分别在N2a、Hepa1-6和NIH3T3三种细胞系中进行CGI-2的CRISPR/Cas9的基因敲除,在验证敲除成功的前提下通过实时定量分析的方法判断在CGI-2缺失的情况下Dlk1-Dio3印记区域内父母本基因的表达水平变化。其结果显示,敲除位点远端的Dio3并没有明显的变化、Dlk1有不显著的下调趋势;位于次远端的Mirg呈现出上调的趋势,但在不同细胞系中其上调程度存在差异;而位于区域中间部分的Gtl2、Rtl1、Rian、Meg8、Irm和AB063319有显著的上调趋势。综上可知,CGI-2的缺失会导致Dlk1-Dio3印记区域中各基因表达水平的变化,即CGI-2对该印记区域的表达具有调控作用。最后,检验在删除CGI-2和未删除CGI-2的两种情况下Dlk1-Dio3印记区域内Dlk1-DMR、Gtl2-DMR、IG-DMR和CGI-1-DMR这四个差异甲基化区域的甲基化状态。结果显示,在删除CGI-2和未删除CGI-2的两种情况这四个差异甲基化区域均为高甲基化状态,即未发生改变。综上所述,CGI-2的缺失会导致其所在的Dlk1-Dio3印记区域内多种基因表达水平发生变化,且这种变化趋势在不同细胞系中大致相同;CGI-2对该印记区域中基因表达的调控作用与该区域内其他差异甲基化位点的甲基化状态无关。
[Abstract]:The Dlk1-Dio3 imprinting gene cluster is located at the end of chromosome 12 in mice and on chromosome 14 in humans.The imprinted region plays an important role in regulating the growth and development of the embryo and placenta, and also affects the metabolism of adults and the function of the brain.The expression of genes in the region showed a dynamic process during growth and development. CGI-2 was located near miR-341 and miR-1188 in the region, which was the first differential methylation region in which the female parent was found to be methylated and the male parent was demethylated.And it is the original of DNA function which binds CTCF and has insulator activity.Therefore, the Dlk1-Dio3 imprinting region was selected as the research object, CRISPR/Cas9 gene editing technique was used to delete the long fragment of CGI-2 genome, and the expression changes of upstream and downstream genes after CGI-2 knockout were analyzed at the cellular level.Firstly, the expression levels of genes in the Dlk1-Dio3 imprinting region of the cell line were analyzed, and three cell lines suitable for knockout test were selected.The three cell lines selected were N2a with higher gene expression level in the Dlk1-Dio3 imprinting region, Hepa1-6 with lower gene expression level in the imprinted region and NIH3T3, a non-cancer cell line.Two pairs of wizards RNAs for CRISPR/Cas9 knockout experiment were designed by guided RNA prediction site, and px330 recombinant plasmid was constructed.Secondly, the CGI-2 knockout pretest was carried out in N2a cells, and the suitable guide for the experiment was selected.Then, the gene knockout of CGI-2 CRISPR/Cas9 was performed in N2aHep Hepa1-6 and NIH3T3 cell lines, and the expression level of parental gene in Dlk1-Dio3 imprinted region was determined by real-time quantitative analysis on the premise of verifying the success of knockout.The results showed that there was no significant change in Dio3 of the distal knockout site, and the down-regulation of Dlk1 was not significant. The Mirg located at the subdistal end showed an up-regulation trend, but there were differences in the degree of upregulation in different cell lines.In the middle part of the region, Gtl2Rtl1, Rtl1, Rtl1, Meg8, Irm and AB063319 showed a significant upward trend.It can be concluded that the absence of CGI-2 will lead to the change of gene expression level in the Dlk1-Dio3 imprinting region, that is, CGI-2 can regulate the expression of the imprinted region.Finally, the methylation status of the four differential methylation regions of the Dlk1-Dio3 imprinted region (Dlk1-DMR-Gtl2-DMR-IG-DMR and CGI-1-DMR) was examined under the condition of deleting CGI-2 and not deleting CGI-2.The results show that the four differential methylation regions are hypermethylated in the case of deleting CGI-2 and not deleting CGI-2, that is, no change has taken place.In conclusion, the absence of CGI-2 may result in changes in the expression levels of multiple genes in the Dlk1-Dio3 imprinting region.The effect of CGI-2 on gene expression in the imprinted region was not related to the methylation status of other differentially methylated sites in different cell lines.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78
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本文编号：1758605