中华绒螯蟹血细胞microRNAs在螺原体感染中的免疫调控功能研究

Haemocytes are major cells in the crustacean cellular defence system. At present, it has become a hot topic in research to probe into the relationship between microRNAs (miRNAs) and immune response for disease control and prevention. This project will be based on our primary screening results of the miRNAs of hemocytes in Chinese mitten crab Eriocheir sinensis infected with a novel pathogen, Spiroplasma eriocheiris. We intend to use this pathogen to make further study on functions of the miRNAs in immune regulation in vivo and vitro. First, with using transcriptome sequencing technology, to screen sufficiently the immune related genes of the crab hemocytes during infection with Spiroplasma eriocheiris; Second, with using bioinformatics, to analyze the hemocyte miRNA transcriptome data from the infected and uninfected groups and to excavate the immune related miRNAs; Third, miRNA chip and gene expression chip will be used to screen and identify the miRNA target genes; Fourth, to detect transcription of the miRNAs and the expression of associated proteins during infection with Spiroplasma eriocheiris by quantitative RT-PCR and Western blot respectively; Fifth, to demonstrate functions of the miRNAs and their relationship with target genes by dual-luciferase reporter gene assay with the miRNAs mimics or inhibitor. Our results will suggest that elucidation of the molecular mechanisms responsible for miRNA regulation of the host's innate immune system and should help with the development of new control strategies to prevent or treat Spiroplasma eriocheiris infections in crustaceans.

血细胞是甲壳动物参与免疫防御的重要细胞。miRNAs与免疫应答间的调控关系是当今热点之一。本项目在前期中华绒螯蟹血细胞miRNAs筛选基础上，拟采用新型水生病原微生物螺原体为致病源，分别在体内外条件下开展血细胞miRNAs在螺原体感染过程中的免疫调控功能研究。首先，利用转录组测序技术，筛选蟹螺原体感染中的免疫相关基因；其次，利用生物信息学手段，对蟹感染螺原体前后血细胞miRNA转录组数据进行分析，挖掘与免疫相关的miRNAs；随后，结合miRNA芯片与基因表达芯片进一步筛选和鉴定免疫miRNAs及其靶基因，再次，分别利用荧光定量RT-PCR和Western blot检测蟹不同感染时期miRNAs转录和相关蛋白表达情况；最后，通过miRNA模拟物和抑制剂，并结合双荧光素酶报告基因测定，初步确定miRNAs功能及其与靶基因的相互调控关系，为诠释虾蟹免疫防御分子调控机理和疾病防治新对策奠定基础。

本项目通过转录组测序、生物信息学分析等方法筛选出中华绒螯蟹螺原体感染中免疫相关基因及miRNA基因以及其靶基因，并针对靶基因进行了验证，挖掘出与免疫相关的 miRNAs，初步揭示了miRNAs 在中华绒螯蟹免疫功能中的作用。首先开展实验验证50个显著差异表达miRNAs的靶基因，完成了通过数据挖掘的117个免疫相关基因的调控miRNA的预测以及靶基因的功能富集和通路分析。然后经过miRNA芯片、基因表达芯片及生物信息学等多种分析方法得到中华绒螯蟹免疫相关基因MyD88和Tube的潜在靶miRNAs，并选取其中可能性最大的6条靶miRNAs进行体内验证实验，最终证明miRNA-7为MyD88的靶miRNA。根据荧光定量PCR的结果分析，miR-7在河蟹肝胰腺组织中表达量最高，其次是鳃和血淋巴组织，在肌肉、肠、心脏和神经中的表达量较少，在眼柄中没有表达。miR-7的表达范围很广泛，预示了miR-7可能担负着较复杂的免疫功能。针对前期筛选的一些免疫相关miRNA基因，合成相应的模拟物和抑制剂，开展其功能的研究。研究发现，两条宿主编码的miRNAs分别作用于宿主Toll信号通路中最重要的接头基因MyD88和Tube的3’UTR区域，从而抑制宿主MyD88和Tube基因的表达。MyD88和Tube基因表达量的降低直接调控下游抗菌肽的表达，包括抗脂多糖因子（Anti-lipopolysaccharide factors，ALFs）、甲壳素（Crustin）、溶菌酶（lysozyme）等。宿主通过自身编码的miRNAs去抑制宿主Toll免疫信号通路，从而达到免疫防御功效。