东北虎胎盘microRNA表达谱、转录组及microRNA-424调节MAPK通路分子机制研究

发布时间：2018-01-25 22:09

本文关键词： 东北虎 microRNA 转录组靶基因 MAPK信号通路表达调控　出处：《东北林业大学》2016年博士论文　论文类型：学位论文

【摘要】：胎盘是哺乳动物妊娠期间由胚胎的胚膜和母体子宫内膜联合长成的母子间交换物质的过渡性器官,具有物质交换、防御、合成和免疫等功能。随着东北虎基因组数据的公布,东北虎表观遗传学成为一个可开拓的前沿领域。MicroRNA是一类由内源基因编码的长度约为18～25个核苷酸(nt)的非编码单链RNA分子,其主要参与基因转录后的表达调控。本研究应用高通量测序技术对东北虎胎盘组织进行microRNA表达谱及转录组分析,并对胎盘组织特异microRNA进行功能验证,得到以下结论：1、利用RNA-seq技术对东北虎胎盘组织进行small RNA测序,去除接头和低质量序列后,将clean reads与东北虎基因组(http://www.ncbi.nlm.nih.gov/genome/?term=tiger)进行比对、注释及表达分析。获得东北虎胎盘组织microRNA表达谱,其中包括465个microRNA,经聚类分析分为41个簇(cluster)和35个家族(family),其中胎盘组织特异性的microRNA有microRNA-424, microRNA-450,microRNA-495, microRNA-376a, microRNA-184, microRNA-369和microRNA-503,利用TAM tools (http://www.cuilab.cn/tam)进行功能分析,其功能主要包括胎儿生长发育、血管生成、骨再生、肌肉发育、凋亡与抗凋亡、细胞增殖与分化、粒细胞生成、免疫反应应答和激素分泌调节等。2、对东北虎胎盘组织转录组进行生物学分析,包括其表达基因的Gene Ontology(GO) 和 Clusters of Orthologous Groups of proteins (COG)分析。其中21103条东北虎基因在GO分析中被注释到45个GO terms,有8462条基因在COG中被注释、分类。利用靶基因预测工具TargetScan和miRanda对靶基因在东北虎胎盘组织转录组上进行预测,获得东北虎胎盘组织microRNA调控靶基因,描述了microRNA靶基因在胎盘组织中生物学功能,同时绘制了microRNA调控靶基因实现胎盘组织功能的分子网络图。3、选取胎盘组织中特异的microRNA,利用Real time PCR、组织原位杂交和Westen blotting分析microRNA-424及其靶基因MAP2K1在东北虎和家猫胎盘组织中的表达,结果显示东北虎胎盘组织中microRNA-424的表达显著低于家猫胎盘组织(**p0.01),而其靶基因MAP2K1的表达在东北虎胎盘组织中显著高于家猫胎盘组织(**p0.01)。4、通过靶位点突变及双荧光素酶分析得出MAP2K13'UTR区域microRNA-424靶位点突变后,荧光素酶活性无显著差异,而未突变组荧光素酶活性显著下调(**p0.01),证实microRNA-424可直接靶向结合MAP2K13'UTR区域,并在东北虎成纤维细胞中过表达microRNA-424后,Westen blotting证实microRNA-424可显著下调MAP2K1的蛋白表达量(**p0.01)。为验证microRNA-424在东北虎成纤维细胞中的生物学功能,Westen blotting评估侵染microRNA-424病毒颗粒后的东北虎成纤维细胞MAP2K1基因下游ERK1/2的磷酸化及蛋白表达量,MTT法评定东北虎成纤维细胞增值能力,结果证实,microRNA-424不但直接调节MAP2K1的转录表达,同时影响下游ERK1/2的表达,从而起到抑制细胞增殖分化的作用。综上所述,microRNA-424及其靶基因MAP2K1在东北虎与家猫胎盘组织中的表达差异可能与猫科动物体型大小有关,该实验结果初步证明东北虎、家猫间的体型大小与microRNA-424的转录后调控相关,但具体调控机制仍需要在动物内体进行基因敲除或突变实验来证实。
[Abstract]:The placenta is a transitional organ exchange substance during pregnancy by mammalian embryo embryo membrane and the endometrium of a joint between mother and child, with material exchange, defense, synthesis and immune functions. With the published tiger genome data, epigenetic studies as a pioneering frontier is a kind of endogenous encoding by.MicroRNA the gene length is about 18 ~ 25 nucleotides (NT) tiger non encoding single stranded RNA molecules, which are involved in the regulation of the expression of gene transcription. After the application of high-throughput sequencing technology on tiger placenta in microRNA expression profiling and transcriptome analysis, and to verify the function of the placenta specific microRNA. Get the following conclusions: 1. The small RNA sequencing of the Siberian tiger placenta using RNA-seq technology to remove the joint and low quality sequences, clean reads and http://ww group (northeast tiger gene W.ncbi.nlm.nih.gov/genome/? Term=tiger) for comparison, analysis and expression of the notes. Get tiger placenta microRNA expression profiles, including 465 microRNA, cluster analysis was divided into 41 clusters (cluster) and 35 (family), the family of placental tissue specific microRNA microRNA-424, microRNA-450, microRNA-495, microRNA-376a, microRNA-184. MicroRNA-369 and microRNA-503, using TAM tools (http://www.cuilab.cn/tam) functional analysis, its function mainly includes fetal growth, angiogenesis, bone regeneration, muscle development, apoptosis and anti apoptosis, cell proliferation and differentiation, cell formation, immune response and regulation of hormone secretion,.2, biological analysis on tiger placenta the transcriptome, including the gene expression of Gene Ontology (GO) and Clusters of Orthologous Groups of proteins (COG) analysis in 211. 03 tiger genes have been annotated to 45 GO terms in the GO analysis, 8462 genes were annotated in COG, TargetScan and miRanda. Classification prediction tools were used to predict the target gene in the placenta of northeast tiger transcriptome using the target gene, obtained tiger placenta microRNA regulated target genes, is described microRNA biological target gene in placenta, and to draw the molecular network diagram.3 microRNA regulated target genes achieve placenta function, select specific placental tissues by Real microRNA, time PCR, Westen blotting and in situ hybridization analysis of the expression of microRNA-424 and its target gene MAP2K1 in the Siberian tiger and cat in the placenta, the the expression of microRNA-424 in placenta of the tiger cats was significantly lower than that of the placenta (**p0.01), and the expression of its target gene MAP2K1 was significantly higher in the Northeast Tiger placenta In the cat placenta (**p0.01) by.4, the target site mutation and dual luciferase analysis MAP2K13'UTR microRNA-424 target mutation, no significant differences in luciferase activity, while no mutation group significantly reduced luciferase activity (**p0.01), confirmed that microRNA-424 can directly target MAP2K13'UTR region, and over expression of microRNA-424 in fibroblasts in Northeast China tiger, Westen blotting confirmed that microRNA-424 could significantly decrease the protein expression of MAP2K1 (**p0.01). In order to verify microRNA-424 to the biological functions of fibroblasts in the Siberian tiger, the Westen assessment of blotting phosphorylation and protein infection of microRNA-424 virus particles after the tiger into downstream of MAP2K1 gene expression of ERK1/2 cells, MTT evaluation of northeast tiger method of fiber cell proliferation. The results confirmed that the expression of microRNA-424 not only directly regulate the MAP2K1, at the same time. The expression of downstream ERK1/2 ring, thus inhibiting the cell proliferation and differentiation. These differences may be related to the size of the feline animal size of microRNA-424 and the expression of target gene MAP2K1 in the Siberian tiger and cat in the placenta, the experimental results proved that the Siberian tiger, the size and the cat microRNA-424 transcription regulation, but the specific mechanisms still need to be in the animal in gene knockout or mutation experiments to confirm.

【学位授予单位】：东北林业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q953

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