禽呼肠孤病毒强毒株和弱毒疫苗株感染鸡肝癌细胞的转录组学分析

摘要

To discover differential expressed key genes in LMH cells infected with ARV virulent and attenuated vaccine strain,RNA-seq sequencing was utilized to perform the transcriptomic analysis.LMH cells were infected with ARV virulent strain S1133 and attenuated strain aS1133 at the dosage of 1 MOI,the infected cells and uninfected cells were collected at 6,12 and 24 hpi to prepare total RNA samples for transcriptomic analysis by Illumina HiSeqTM 2000 sequencer.Genes between S1133-infected and aS1133 infected cells with expression difference times ≥-2 and P≤0.001 were set as differentially expressed genes for further analysis.The analysis results showed that compared with aS1133-infected cells,4 013 differentially expressed genes (DEGs) were discovered,including 58 up-regulated and 5 down-regulated DEGs at 6 hpi;1 429 up-regulated and 354 down-regulated at 12 hpi;1 680 up-regulated and 481 down-regulated at 24 hpi.These DEGs expression modes were categorized into 10 co-expression trends.With GO functional annotation analysis,the biological functions of these DEGs were associated with GTPase regulator activity,signal transducer activity,protein kinase activity;and these DEGs were involved in response to biotic stimulus,cell apoptotic process,biological regulation,signal transduction.The pathway analysis showed that DEGS were involved in a serious of signal pathways such as Toll-like receptor signal pathway,TGF-β signal pathway.The results of qRT-PCR for detection of DEGs randomly selected were consistent with those of transcriptomic analysis.The above data compared and analyzed that at the transcription level,differentially expressed genes in LMH cells after ARV virulent and attenuated strain infection and explored the possible different responses for host cells to ARV different strains,which would contribute to further understand ARV pathogenesis.%本研究旨在研究禽呼肠孤病毒(avian reovirus,ARV)强毒株S1133和弱毒疫苗株aS1133分别感染鸡肝癌(LMH)细胞后,LMH细胞基因组的转录水平变化.将1 MOI的ARV强毒株S1133和弱毒疫苗株aS1133分别感染LMH细胞,感染后6、12和24 h,收集感染细胞和阴性对照细胞,制备总RNA样品,使用Illumina HiSeqTM 2000测序仪进行转录组学测序.以感染组之间样品中上下调转录差异≥2且P≤0.001的基因为差异基因,进行生物信息学分析并进行荧光定量PCR验证.分析结果表明,与ARVaS1133感染组相比,S1133感染组共有4 013个差异表达基因,其中6 hpi时,58个上调表达差异基因,5个下调基因;12 hpi时,1 429个上调基因,354个下调基因;24 hpi时,1 680个上调基因,481个下调基因.K-平均值聚类分析结果说明这些差异基因的表达模式主要呈上调表达模式,并且有10种共表达趋势.GO功能注释和分析结果表明差异基因主要具有GTP酶调节活性、信号转导活性、蛋白激酶活性等生物功能;主要参与机体应激、细胞信号转导、细胞凋亡等生物过程.Pathway富集分析结果表明差异表达基因主要参与Toll样受体信号通路、TGF-β信号通路、病原体感染应答等细胞信号通路.随机选择部分差异表达基因进行荧光定量RT-PCR验证,其结果表明与转录组学测序结果基本一致.以上数据比较分析了ARV强毒株和弱毒疫苗株感染后LMH细胞的基因表达的变化差异,探讨了宿主细胞对ARV不同毒力毒株的可能不同应答机制,有利于进一步阐明ARV致病机制.