Jiangsu Key Laboratory for Biodiversity & Biotechnology, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China.
Fish Shellfish Immunol. 2012 Feb;32(2):345-52. doi: 10.1016/j.fsi.2011.11.027. Epub 2011 Dec 6.
The Chinese mitten crab Eriocheir sinensis is one of the most important freshwater aquaculture crustacean species in China. MicroRNAs (miRNAs) are small non-coding RNAs that are important effectors in the intricate host-pathogen interaction network. To increase the repertoire of miRNAs characterized in crustaceans and to examine the relationship between host miRNA expression and pathogen infection, we used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from haemocytes of E. sinensis under normal conditions and during infection with Spiroplasma eriocheiris. The high-throughput sequencing resulted in approximately 30,975,151 and 30,826,277 raw reads corresponding to 12,077,088 and 16,271,545 high-quality mappable reads for the normal and infected haemocyte samples, respectively. Bioinformatic analyses identified 735 unique miRNAs, including 36 that are conserved in crustaceans, 134 that are novel to crabs but are present in other arthropods (PN-type), and 565 that are completely new (PC-type). Two hundred twenty-eight unique miRNAs displayed significant differential expression between the normal and infected haemocyte samples (p < 0.0001). Of these, 133 (58%) were significantly up-regulated and 95 (42%) were significantly down-regulated upon challenge with S. eriocheiris. Real-time quantitative PCR (RT-qPCR) experiments were preformed for 10 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first report of comprehensive identification of E. sinensis miRNAs and of expression analysis of E. sinensis miRNAs after exposure to S. eriocheiris. Many miRNAs were differentially regulated when exposed to the pathogen, and these findings support the hypothesis that certain miRNAs might be essential in host-pathogen interactions. Our results suggest that elucidation of the molecular mechanisms responsible for miRNA regulation of the host's innate immune system should help with the development of new control strategies to prevent or treat S. eriocheiris infections in crustaceans.
中华绒螯蟹（Eriocheir sinensis）是中国最重要的淡水养殖甲壳类动物之一。microRNAs（miRNAs）是在复杂的宿主-病原体相互作用网络中起重要作用的小非编码 RNA。为了增加甲壳类动物中已鉴定的 miRNA 谱，并研究宿主 miRNA 表达与病原体感染之间的关系，我们使用 Illumina/Solexa 高通量测序技术对正常条件下和感染螺旋体（Spiroplasma eriocheiris）时中华绒螯蟹血细胞中的两个小 RNA 文库进行了测序。高通量测序产生了大约 30,975,151 和 30,826,277 个原始读数，分别对应于正常和感染血细胞样本的 12,077,088 和 16,271,545 个高质量可映射读数。生物信息学分析鉴定出 735 个独特的 miRNAs，其中 36 个在甲壳类动物中保守，134 个在螃蟹中是新的，但在其他节肢动物中存在（PN 型），565 个是完全新的（PC 型）。228 个独特的 miRNAs 在正常和感染的血细胞样本之间表现出显著的差异表达（p<0.0001）。其中，133 个（58%）显著上调，95 个（42%）显著下调。实时定量 PCR（RT-qPCR）实验对两个样本中的 10 个 miRNAs 进行了检测，测序和 RT-qPCR 数据之间存在一致性。据我们所知，这是首次全面鉴定中华绒螯蟹 miRNAs 并分析中华绒螯蟹在暴露于螺旋体后 miRNAs 的表达情况。当暴露于病原体时，许多 miRNAs 被差异调节，这些发现支持某些 miRNAs 在宿主-病原体相互作用中可能是必不可少的假说。我们的结果表明，阐明负责宿主先天免疫系统的 miRNA 调节的分子机制，应该有助于开发新的控制策略，以预防或治疗甲壳类动物中的螺旋体感染。
