作 者：
罗金;袁小松;郝佳伟;田占成;谢俊仁;陈泽;任巧云;殷宏;罗建勋;刘光远
关键词：
亚洲璃眼蜱;微小RNA （microRNA）;miR-451;巨噬细胞抑制游走因子;MIF
摘 要：
【目的】microRNA （miRNA）作为一类内源性的非编码RNA，仅有18-25 nt，其广泛存在于动植物细胞中,可诱导生物基因沉默，参与细胞生长、发育、基因转录和翻译等诸多生命活动的调控过程。以亚洲璃眼蜱为研究对象，对miR-451及其靶基因（巨噬细胞游走因子， MIF）在相互作用关系进行分析。【方法】参考miR-451成熟体序列（AAA CCG UUA CCA UUA CUG AGU UU）来自研究单元亚洲璃眼蜱高通量测序所获得的结果。根据该成熟体序列设计stem-loop 及PCR 引物，RT-PCR 扩增获得蜱源性miR-451序列；并对不同物种来源的miR-451序列特征进行分析。基因合成方法获得抑制miR-451的dsRNA 序列，用于亚洲璃眼蜱饥饿成蜱体内注射。参考美洲钝眼蜱MIF 基因（登录号：AF289543.2），设计亚洲璃眼蜱的特异性实时荧光定量引物，以β-actin 作为内参基因，采用SYBR Green real-time RT-PCR 方法检测注射dsRNA 后亚洲璃眼蜱饥饿成蜱不同发育时间点MIF 基因的表达水平，以及miR-451的表达谱特征。【结果】琼脂糖凝胶电泳检测miR-451的PCR 产物条带与预期大小一致，为72 nt。不同物种来源的miR-451具有较高的保守性，特别是种子序列极度保守，仅在短尾负鼠miR-451成熟体的19位点处存在A→U的突变。miR-451的RNA 干扰实验证实，0-30 h miR-451表达逐渐上调，6 h 达到最高值，其拷贝数为1.0×10~8。随后表达丰度逐渐降低。30 h 其表达水平与PBS 对照组相同。48-60 h miR-451再次出现一个表达上调微小变化过程。而miR-451抑制后的MIF 直到30 h 才有表达的上调，42 h 达到峰值（拷贝量仅为9.0×10~3），此后其表达规模受到抑制，48 h 时与PBS 对照组水平一致。84 h 后表达才逐渐上调，直到90 h 达到峰值，并呈现正态分布趋势。【结论】利用RT-PCR 获得亚洲璃眼蜱成蜱阶段miR-451序列，生物信息学方法分析了miR-451序列在不同物种间具有较高保守性。miRNA的保守性决定其靶标基因的特异性，因此，以上结果提示miR-451在动物细胞中可能扮演重要的生物学功能，是MIF 功能发挥的一个重要调控因子。MIF 作为miR-451靶标基因，其功能的实现可能受该miRNA的调控。基于此类推测qPCR 及RNA 干扰分析表明，miR-451参与了MIF的表达调控。且是一种负调控作用。当miR-451表达量升高时，MIF 表达量明显下调。此研究首次证实miR-451在亚洲璃眼蜱成蜱发育阶段的表达是一种普遍现象，且对MIF 存在负调控作用。这一研究为后期miR-451参与蜱的免疫应答及miR-451与靶标基因的相互作用机制提供了参考。同时证明，miRNA 参与基因功能的调控具有一定的特异性和时序性。
译 名：
Dynamic Analysis of Expression Level of miR-451 and MIF Gene in Different Developmental Stages of Adult Hyalomma asiaticum
作 者：
罗金(1);;袁小松(1);;郝佳伟(1);;田占成(1);;谢俊仁(1);;陈泽(1);;任巧云(1);;殷宏(1);;罗建勋(1);;刘光远(1)
单 位：
(1)中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点实验室;;;;甘肃省动物寄生虫病重点实验室, 兰州, 730046
摘 要：
【Objective】The microRNAs(miRNAs) are a class of small single-stranded noncoding RNAs(18-25 nt) in both unicellular and multicellular organisms. Previous studies showed that miRNAs play pivotal roles in regulating diverse developmental processes by targeting mRNAs for translational repression, development, or transcription. To dissect the interaction between miR-451 and target gene(Macrophage Migration Inhibitory Factor, MIF), Hyalomma asiaticum ticks were analyzed. 【Method】The primers were designed for stem-loop and PCR based on the mature sequence of miR-451（AAA CCG UUA CCA UUA CUG AGU UU）from High-through sequence results of Hyalomma asiaticum. The mature sequence of miR-451 was amplified by RT-PCR and analyzed miR-451 sequences from different species. The dsRNA of miR-451 was obtained by gene synthesis and it was injected into unfed-adult H. asiaticum. Referencing the MIF gene of Amblyomma americanum in GenBank(Accession number: AF289543.2) for specific primers of qPCR to H. asiaticum, and the β-actin gene as an internal reference gene. After injecting dsRNA of miR-451, the MIF and miR-451 expression level of various developmental stages of unfed-adult ticks were detected by SYBR Green real-time RT-PCR. 【Result】The miR-451 PCR product was consistent with the prediction that it is 72 nt by agarose gel electrophoresis. The result showed miR-451 has a high conservatism in different species by multiple sequence alignment analysis, only Monodelphis domestica has a base mutations(A→U) at 19 sites. Our results showed that the miR-451up-regulation at 0-30h and 6h had highest expression level with a copy number of 1.0×10~8. Subsequently, the expression level gradually reduced, such that they had same level at 30 h with PBS control. The expression had an up-regulation until 48-60 h again. After the miR-451 was restrained the MIF was up-regulated at 30 h, and 42 h peaks(with copy levels of 9.0×10~3), since the expression is restrained, and the level is a same with PBS control at 48. At 84 h the expression level of MIF was up-regulated and the peak was at 90 h. All the expression levels showed a normal distribution. 【Conclusion】 In this paper, the miR-451 sequence was amplified by RT-PCR, and we showed that miR-451 had high conservatism across different species. The conservatism of miR-451determined the target gene specificity. These results suggested that miR-451 plays an important biological function in animal cells and is an important regulatory factors for MIF function. As a target gene of miR-451 functions of MIF was regulated by miR-451using the RNAi, and they are reverse regulated. When the expression level of miR-451 is up-regulated, MIF expression were significantly lowered. The study is the first to confirm that expression of miR-451 is a common phenomenon in developmental stages of H. asiaticum ticks and that miR-451 has a negative regulatory effect on MIF. This study provides a reference for miR-451participation in immune response of ticks and the interaction mechanism between miR-451and target genes. It also confirms that the miRNA regulatory behavior has specificity and offers scheduling for gene function.
