长链非编码RNA在脂多糖刺激的大鼠肠巨噬细胞中表达谱分析

发布时间：2018-04-24 07:51

  本文选题：长链非编码RNA + 肠巨噬细胞　； 参考：《江苏大学》2017年硕士论文

【摘要】：目的本课题旨在分析长链非编码RNA(long noncoding RNA,lncRNA)在脂多糖(Lipopolysaccharide,LPS)诱导大鼠的肠巨噬细胞组与未处理组之间差异表达谱,并对差异表达的mRNA进行生物信息学分析,为初步探讨lncRNA的调控机制奠定基础。方法1.提取六只大鼠的肠巨噬细胞进行培养,随机分成两组,实验组加脂多糖(浓度为1 mg/L)诱导大鼠的肠巨噬细胞,对照组则未加脂多糖。应用高通量基因芯片技术(Affymetrix Gene Chip Mouse Transcriptome Array 1.0)检测两组之间差异表达的lncRNA和mRNA,并用分层聚类的方法分析两组间差异表达的lncRNA和mRNA。2.对芯片筛选出差异表达的mRNA进行基因功能分析(GO-Analysis)和信号通路分析(Pathway-Analysis)探索其参与的生物学功能,并构建差异表达lncRNA和mRNA的共表达网络,从中发现处于核心调控地位的lncRNA。3.结合共表达网络分析结果和基因芯片数据,挑选出差异表达的lncRNA和mRNA采用实时荧光定量PCR技术,对芯片结果进行验证。结果1.将差异表达的纳入标准设为:差异表达倍数(Flod-change)1.5,P值0.05,与对照组相比,LPS处理的肠巨噬细胞组lncRNA和mRNA表达谱发生明显变化,差异表达的lncRNA共有357个,其中上调的有245个,下调的有112个。差异表达的mRNA共有542个,其中上调的有187个,下调的有355个。2.基因功能分析显示表达上调的m RNA主要涉及炎症反应、固有免疫应答、代谢过程、信号转导等生物学功能;表达下调的mRNA主要涉及代谢过程、细胞周期、免疫应答以及凋亡过程等生物学功能。通路分析显示与上调基因相关的显著性通路主要包括NF-kappa B信号通路、B细胞受体信号通路、细胞凋亡信号通路等;与下调基因相关的显著性通路主要有代谢通路、磷脂酰肌醇信号通路、细胞因子-细胞因子受体相互作用以及Toll样受体信号通路等。构建lncRNA和mRNA共表达网络,其中lncRNA NONMMUT062258、NONMMUT046238、NONMMUT024673、NONMMUT062258处于核心地位。3.实时荧光定量PCR结果显示差异表达的lncRNA和mRNA与基因芯片数据一致。结论本研究首次通过基因芯片技术对LPS处理的大鼠肠巨噬细胞中lncRNA的表达谱进行了检测。芯片结果显示与无LPS处理的对照组相比,lncRNA在LPS处理的肠巨噬细胞中呈差异表达。实时定量PCR验证结果证实芯片数据稳定而可靠。因此差异表达的lncRNA可能参与调控LPS诱导肠巨噬细胞引起的炎症反应。此外用生物信息学分析探讨了潜在的调控机制。尽管需要更多的研究来证明这些lncRNA的确切调控机制,但我们鉴定的基因组数据可以为深入研究肠屏障功能障碍时肠巨噬细胞调控炎症反应的分子机制提供新的线索和思路。
[Abstract]:Objective to analyze the differential expression profiles of long-chain noncoding RNA(long noncoding RNAs in lipopolysaccharide polysaccharide (LPS) -induced intestinal macrophage group and untreated group, and to analyze the differentially expressed mRNA by bioinformatics. It lays a foundation for the preliminary study of the regulatory mechanism of lncRNA. Method 1. The intestinal macrophages of six rats were cultured and randomly divided into two groups. The experimental group was induced by lipopolysaccharide (1 mg / L), while the control group was not lipopolysaccharide. The differentially expressed lncRNA and mRNAs were detected by affymetrix Gene Chip Mouse Transcriptome Array 1.0, and the lncRNA and mRNA.2differentially expressed between the two groups were analyzed by hierarchical clustering. Gene function analysis (GO-analysis) and signal pathway analysis (Pathway-Analysis) of differentially expressed mRNA were carried out to explore its biological function, and to construct coexpression network of differentially expressed lncRNA and mRNA, from which we found lncRNA.3in the core regulatory position. Based on the results of coexpression network analysis and gene chip data, the differentially expressed lncRNA and mRNA were selected and verified by real-time fluorescence quantitative PCR. Result 1. The inclusion criteria of differential expression were as follows: the differential expression multiple of lncRNA and mRNA in LPS-treated intestinal macrophages was 0.05. The expression profiles of lncRNA and mRNA in LPS-treated intestinal macrophages were significantly changed, and there were 357 differentially expressed lncRNA, 245 of which were up-regulated. There were 112 downgrades. Of the 542 differentially expressed mRNA, 187 were up-regulated and 355. 2. Gene function analysis showed that the up-regulated expression of m RNA was mainly related to inflammatory response, innate immune response, metabolic process, signal transduction and other biological functions, while down-regulated mRNA was mainly involved in metabolic process, cell cycle, and so on. Immune response and apoptosis process and other biological functions. Pathway analysis showed that significant pathway related to up-regulation gene mainly included NF-kappa B signal pathway, cell apoptosis signal pathway and so on, and the significant pathway related to down-regulation gene was metabolic pathway. Phosphatidylinositol signaling pathway, cytokine-cytokine receptor interaction and Toll-like receptor signaling pathway. The coexpression network of lncRNA and mRNA was constructed, in which lncRNA NONMMUT062258, NONMMUT046238, NONMMUT024673, NONMMUT062258 was at the core. The results of real-time fluorescence quantitative PCR showed that the differentially expressed lncRNA and mRNA were consistent with the gene chip data. Conclusion for the first time, the expression profile of lncRNA in LPS treated rat intestinal macrophages was detected by gene chip technique. The microarray results showed that there was a difference in the expression of lncRNA in LPS treated intestinal macrophages compared with the control group without LPS treatment. Real-time quantitative PCR verification results show that the chip data is stable and reliable. Therefore, differentially expressed lncRNA may be involved in regulating the inflammatory response induced by LPS in intestinal macrophages. In addition, the potential regulation mechanism was analyzed by bioinformatics. Although more studies are needed to prove the exact regulatory mechanisms of these lncRNA, the genomic data we have identified can provide new clues and ideas for the further study of the molecular mechanisms of intestinal macrophages regulating inflammatory response in the context of intestinal barrier dysfunction.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R57

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本文编号：1795780