转基因小鼠皮肤转录组测序揭示microRNA-137的毛色生成调控机制

发布时间：2018-05-04 09:23

本文选题：转基因小鼠 + microRNA-137　；参考：《山西农业大学》2016年博士论文

【摘要】：毛色生成调控机制的研究既可以帮助我们进一步了解哺乳动物生物学特征的发生机制,又可以促进天然毛色在毛用产品工业的应用,避免由于染色对人们健康的危害,具有重要的研究意义。microRNA (miRNA)通过与其靶基因的3’UTR端的结合位点相结合,抑制或降解靶基因的表达,从而参与多种生物学过程的调控。在之前的研究中,我们成功地构建了包括灰色和棕色毛色表型在内的microRNA-137 (miR-137)转基因小鼠,并发现在不同毛色转基因小鼠的皮肤中,miR-137的表达量差异显著。然而,miR-137导致转基因小鼠发生毛色表型变化的毛色生成调控机制仍尚未研究。为了进一步研究miR-137的毛色生成调控机制,本文通过对灰色、棕色miR-137转基因小鼠背部皮肤进行转录组测序,发现miR-137不同程度过表达导致小鼠毛色变化的过程中所调控的基因以及基因功能、影响的信号通路和生物学过程；利用对小鼠皮肤组织的HE染色和免疫荧光实验验证了上述信息学分析所提出的miR-137可以影响黑色素细胞内黑色素合成过程这一假设；通过生物信息学预测和双荧光报告实验验证,寻找miR-137导致小鼠毛色变化的过程中直接调控的毛色相关靶基因,同时在小鼠皮肤黑色素细胞中过表达miR-137,观察其靶基因和靶基因毛色通路中下游基因的表达变化以及黑色素生成量的变化,揭示了miR-137调控毛色生成的作用机制。1.以灰色、棕色miR-137转基因小鼠皮肤作为实验材料,通过转录组测序技术,检测到两种毛色小鼠皮肤中表达的基因约19,000个。利用这些基因在不同毛色小鼠皮肤中的表达量差异,我们筛选出93个表达量差异程度在2倍以上且符合统计学意义(0.8≤Diverse probability≤1)的表达量显著差异的基因(差异基因)。为了验证转录组测序结果,我们通过荧光定量实验对差异基因在灰色、棕色转基因小鼠皮肤中的相对表达量进行实验验证,结果显示所选基因的表达量趋势与测序结果一致,这证明了此次转录组测序数据的可靠性,同时为进一步对表达基因为基础的后续分析奠定了重要的基础。2.为了进一步了解miR-137调控两种毛色小鼠表型产生的过程中所影响的基因功能和信号通路,我们首先利用Protein ANalysis THrough Evolutionary Relationships (PANTHER)软件对所有差异基因的基因功能进行Go Ontology (GO)功能注释并观察差异基因在GO功能中的宏观分布。继而,通过Database for Annotation, Visualizationand Integrated Discovery (DAVID)软件,对已经注释GO功能的差异基因进行GO功能富集,结果发现差异基因显著富集于黑色素合成功能。然后,我们将差异基因富集到Kyoto Encyclopedia of Genes and Genomes (KEGG)数据库中的信号通路中,发现差异基因显著富集于黑色素合成这一通路。从而分析出,miR-137在黑色素细胞内的黑色素合成过程中起到关键的调控作用,导致了不同毛色小鼠表型的产生。3.为了进一步探究miR-137在小鼠体内过表达后从整体水平上调控小鼠体内的生物学过程变化,我们通过GSEA分析手段,分析灰色、棕色小鼠皮肤转录组测序得到的全部表达基因所共同富集的生物学过程。为了保证分析的准确性,我们选择GSEA数据库中收录的已验证基因功能的Hallmark基因集对转录组获得的全部表达基因进行生物学过程富集分析。同时,为了进一步验证上一章中我们对所选差异基因进行功能富集分析结果的准确性,我们在选取Hallmark基因集的同时也选取GO gene sets基因集进行GSEA分析。从分析结果中我们发现,miR-137在棕色小鼠体内过表达确实引起了黑色素合成的变化,这与上一章所分析的结果一致,并发现miR-137不同程度的表达趋势可以引起小鼠体内不同生物学过程的变化：在灰色小鼠表型中,miR-137通过调控Myc下游基因和参与核糖体相关过程可以调控色素沉着更显著的变化。4.在灰色、棕色小鼠体内表达量差异的基因中对miR-137的靶基因进行生物学预测,并找出可能导致毛色直接变化的靶基因Kit和Myo7a 。结合第二、三章的分析结果中,miR-137在黑色素合成过程中影响了黑色素生成量的变化导致了棕色小鼠表型产生,从而得知miR-137可能通过影响SCF/Kit通路及下游黑色素合成相关基因而导致了棕色毛色小鼠表型的产生。5.利用HE染色和免疫荧光实验,发现miR-137在转基因小鼠过表达引起了黑色素细胞中黑色素生成量的改变。鉴于此,通过双荧光报告实验验证了Kit是miR-137的靶基因,并发现miR-137在小鼠黑色素细胞中过表达可以引起Kit及其下游基因Tyr, Tyrp1和Dct (Tyrp2)的表达量发生变化,调控小鼠皮肤黑色素细胞中黑色素含量的生成。结论： (1)miR-137主要通过影响黑色素细胞中黑色素合成过程导致转基因小鼠浅色表型产生。 (2)miR-137产生灰色、棕色两种转基因小鼠表型的作用机制是不同的。miR-137通过直接影响黑色素细胞中黑色素合成过程导致棕色小鼠表型产生,并且miR-137通过调控Myc下游基因及核糖体相关过程,导致更明显的灰色小鼠表型产生。(3)Kit和Myo7a是miR-137导致转基因小鼠毛色表型变化过程中直接调控的毛色相关靶基因。 (4)通过实验验证了上述分析提出的miR-137过表达影响黑色素细胞中黑色素合成过程这一作用机制的假设以及Kit与miR-137之间的靶向关系,并发现miR-137通过影响SCF/Kit通路中Kit及Kit下游参与黑色素合成过程的主效基因Tvr.Tyrp1和Dct (Tyrp2)的表达抑制黑色素细胞中黑色素的生成。
[Abstract]:The study of the regulation mechanism of hair color generation can help us not only understand the mechanism of mammalian biological characteristics, but also promote the application of natural hair color in wool products industry, and avoid the harm of dyeing to people's health. It has important research significance.MicroRNA (miRNA) through the 3 'UTR end of its target gene. In the previous study, we successfully constructed microRNA-137 (miR-137) transgenic mice, including gray and brown hair color phenotypes, and found the difference in miR-137 expression in the skin of different hair color transgenic mice. In order to further study the regulation mechanism of the hair color generation of miR-137, the transcriptional sequence of the back skin of the brown miR-137 transgenic mice was sequenced to find that the expression of miR-137 in different degrees led to the hair of mouse hair, in order to further study the regulation mechanism of hair color generation in transgenic mice. The signal pathways and biological processes regulated by genes and gene functions during the process of color change; the hypothesis that the miR-137 can affect melanin synthesis in melanocytes by the HE staining and immunofluorescence experiments on the skin of mice is used to predict the process of melanin synthesis in melanocytes; by Bioinformatics The test and double fluorescence report test proved that the hair color related target gene was directly regulated by miR-137 in the process of hair color change in mice, and miR-137 was overexpressed in the skin melanocytes of mice. The changes of the expression of the target gene and the target gene hair color pathway and the change of the melanin formation were observed, and miR-137 was revealed. The mechanism of regulation of hair color generation.1. uses grey, brown miR-137 transgenic mice skin as experimental material and about 19000 genes expressed in the skin of two hair color mice by transcriptional sequencing technology. The differences in the expression of these genes in the skin of different hair color mice were used to screen 93 differences in expression levels. In order to verify the sequencing results of the transcriptional group, we tested the expression of the differential genes in the gray and brown transgenic mice in order to verify the sequence of the transcriptional sequences, which were 2 times more than 2 times and conformed to the significant difference (0.8 < < Diverse probability < 1). The expression trend is consistent with the sequencing results, which proves the reliability of the transcriptional sequence data and lays an important foundation for further analysis of the gene based follow-up analysis,.2. in order to further understand the gene function and signal pathways affected by the miR-137 regulation of the phenotype of two hair color mice. The Protein ANalysis THrough Evolutionary Relationships (PANTHER) software was used to carry out Go Ontology (GO) functional annotations for all the gene functions of the differential genes and to observe the macro distribution of the differential genes in the GO function. The difference gene of GO function was enriched by GO function, and the difference gene was found to be enriched in the melanin synthesis function. Then, we enriched the differential genes into the signaling pathway in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and found that the differential genes were enriched in the melanin synthesis pathway. MiR-137 plays a key regulatory role in melanin synthesis in melanocytes, leading to the production of.3. in different hair color mice in order to further explore the biological process changes in mice from the overall level of miR-137 after overexpression in mice. We analyzed the gray and brown mice by means of GSEA analysis. In order to ensure the accuracy of the analysis, we choose the Hallmark gene set in the GSEA database to carry out the biological process enrichment analysis of all the expressed genes obtained by the transcriptional group. In this chapter, the accuracy of the functional enrichment analysis of the selected differentially differentially expressed genes was obtained. We selected the Hallmark gene set at the same time as the GO gene sets gene set for GSEA analysis. From the analysis, we found that the overexpression of miR-137 in brown mice did cause changes in melanin synthesis, which was analyzed in the previous chapter. The results are consistent, and it is found that different degrees of expression of miR-137 can cause changes in different biological processes in mice: in the phenotype of gray mice, miR-137 can regulate the more significant changes in pigmentation by regulating the downstream genes of Myc and involved in ribosome related processes, and the genes of.4. in gray and brown mice are different in expression. The target gene of miR-137 is predicted by biology, and the target gene Kit and Myo7a that may lead to direct color changes are found. In the analysis of second, third chapters, miR-137 affects the change of melanin production in the melanin synthesis process, resulting in the phenotype production of brown mice, thus knowing that miR-137 may be affected by the influence of SCF/Kit. The pathway and the downstream melanin synthesis related genes led to the production of.5. in brown color mice by HE staining and immunofluorescence experiments. It was found that the overexpression of miR-137 in transgenic mice resulted in the change of melanin formation in melanocytes. In view of this, the target gene of miR-137 was verified by the double fluorescence Report. The overexpression of miR-137 in mouse melanocytes can cause changes in the expression of Kit and its downstream genes, Tyr, Tyrp1 and Dct (Tyrp2), and regulate the formation of melanin in murine skin melanocytes. Conclusion: (1) miR-137 mainly affects the light color phenotype of transgenic mice mainly by affecting melanin synthesis in melanocytes. (2) miR-137 produces grey and brown two transgenic mice, the mechanism of the action is that different.MiR-137 can lead to the phenotypic production of brown mice by directly affecting melanin synthesis in melanocytes, and miR-137 leads to more obvious phenotypes of grey mice by regulating the downstream genes and ribosome related processes of Myc. (3) K It and Myo7a are the hair color related target genes directly regulated by miR-137 in the hair color phenotype changes of transgenic mice. (4) the hypothesis of the effect of miR-137 overexpression on melanin synthesis process in melanocytes and the targeting relationship between Kit and miR-137, and the discovery of miR-137 through experiments were verified by experiments. The expression of major genes, Tvr.Tyrp1 and Dct (Tyrp2), involved in the process of melanin synthesis in the downstream of the SCF/Kit pathway, inhibits the formation of melanin in melanocytes.

【学位授予单位】：山西农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.2

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