转录组测序分析细粒棘球绦虫原头蚴刺激后巨噬细胞Raw264.7 lncRNA表达的研究

Transcriptome sequencing analysis of IncRNA expression in macrophages Raw264.7 stimulated by Echinococcus granulosus protoscoleces

为了解巨噬细胞Raw264.7在应对细粒棘球绦虫原头蚴(PSC)刺激时其长链非编码RNA(lncRNA)的差异表达规律,本研究采用Illumina Hiseq 3000高通量RNA sequencing测序技术对PSC处理以及PBS处理的Raw264.7细胞进行lncRNA测序和生物信息学分析后,进一步采用GO和KEGG数据库分别对差异表达的lncRNAs进行富集分析。结果显示,和PBS对照组相比,PSC处理组有60个lncRNA分子的表达出现显著差异变化,其中17个表达上调,43个表达下调。GO分析结果显示,差异表达的lncRNA分子的靶基因主要与钠离子跨膜转运体活性调节,2型免疫反应正向调节以及自然杀伤细胞凝集素样受体结合等相关;KEGG分析结果显示,差异表达的lncRNA分子的靶基因主要参与VEGF信号、Toll样受体信号通路、NOD样受体等信号通路。本研究进一步分析了与Raw264.7细胞固有免疫相关的lncRNA分子,结果发现有11个模式识别受体(PRR)相关的lncRNA分子。随机选取6个差异表达的lncRNA分子进行了qRT-PCR验证,结果显示其表达趋势与lncRNA测序结果一致。本研究首次系统解析了巨噬细胞Raw264.7应对PSC刺激时其lncRNA的差异表达谱特性,为进一步解析lncRNA在巨噬细胞Raw264.7应对PSC刺激时的免疫调控机制奠定了基础。

To identify the differency of lnc RNAs expression in Raw264.7 cells in response to the stimulation of the Echinococcus granulosus protoscoleces(PSC), the expression of lnc RNAs in the PSC treated-Raw264.7 cells analysed by using Illumina Hiseq 3000 high-through put RNA sequencing technique. The lnc RNA sequeces were then analysed through Gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment. The results showed that the expression of 60 lnc RNAs significantly changed in the PSC-treated group, of which 17 were up-regulated and 43 were downregulated comparing with the control group. GO analysis showed that the target genes of the differentially-expressed lnc RNAs were mainly related to the regulation of sodium ion transmembrane transporter activity, positive regulation of type 2 immune response,and binding of natural killer cell lectin-like receptor. KEGG analysis results showed that the target genes of the differentiallyexpressed lnc RNAs were mainly involved in signal pathways such as VEGF, Toll-like receptor and NOD-like receptor signaling pathways. The present study further analyzed the lnc RNA molecules related to the innate immunity of Raw264.7 cells, and found11 lnc RNA molecules related with the pattern recognition receptor(PRR). At the same time, the expression of 6 differentiallyexpressed lnc RNAs were selected and verified furtherly with q RT-PCR. The results showed that the expression patterns the lnc RNAs were consistent with the RNA-seq results. This study systematically analyzed the expression profile of lnc RNAs in macrophage Raw264.7 against PSC stimulation, and laid the foundation for understanding the immune regulation mechanism of lnc RNAs in the PSC-stimulated macrophage.