Abstract: 【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 1.0×10⁸. 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 9.0×10³), 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.

Key words: Hyalomma asiaticum, microRNA, miR-451, macrophage migration inhibitory factor, MIF