半滑舌鳎(Cynoglossus semilaevis) miR-223的表达特征及免疫应答分析

microRNA(miRNA)是一类内源性、长度约为22个核苷酸的非编码小单链RNA分子，由具有发夹结构的70?90个碱基的单链RNA前体经过Dicer酶加工而成。本研究使用荧光实时定量PCR (Quantitative Real-Time PCR, qRT-PCR)方法，研究了miRNA-223(miR-223)在半滑舌鳎(Cynoglossus semilaevis)各健康组织、鳗弧菌(Vibrio anguillarum)感染后各时间点的免疫组织以及不同病原类似物刺激后头肾细胞中的表达模式。结果显示，miR-223在半滑舌鳎各组织中均有表达，在头肾中表达量最高，在脑和血液中的表达量极低。鳗弧菌感染3组样品4种免疫组织，miR-223表达变化显著，感染后20 h内，鳗弧菌诱导miR-223上调表达。鳗弧菌感染后半滑舌鳎免疫组织miR-223表达变化规律显示，鳗弧菌感染2、6、12、24、48、72、96和168 h后，miR-223在肝、肠、脾、头肾4种组织中出现差异表达。其中，miR-223在半滑舌鳎肝、脾、头肾中表达上调，在肠中表达下调。用LPS、poly I:C、PGN、RGNNV感染半滑舌鳎头肾细胞后，发现经LPS、RGNNV诱导后miR-223上调表达，poly I:C、PGN诱导后miR-223下调表达。研究结果表明，miR-223参与了半滑舌鳎免疫应答过程。本研究结果有助于了解miRNA在半滑舌鳎对病原刺激免疫应答过程中的作用以及半滑舌鳎与病原相互作用中miRNA参与调控的机制。

The Expression Pattern of miR-223 in Different Tissues of Cynoglossus semilaevis and the Regulation of Expression in Response to Infections

MicroRNAs (miRNAs) are a type of endogenous small single-stranded noncoding RNAs that contain approximately 22 nucleotides (nt). miRNAs are the products of single-stranded pre-mRNAs which have 70-90 nt and a stem-loop structure, and spliced by dicer enzyme. Half-smooth tongue sole (Cynoglossus semilaevis) is an important economic marine fish species. The cloning of C. semilaevis miR-223 precursor was predicted to have a typical hairpin structure, which suggested that the precursor sequence we obtained was reliable. To investigate the role of immune related miRNAs in C. semilaevis, we applied quantitative real-time PCR (qRT-PCR) and studied the expression patterns of miR-223 in different tissues of healthy C. semilaevis. miR-223 was expressed in all tested tissues including head kidney, spleen, gill, intestine, ovary, muscle, stomach, liver, skin, blood, and brain. The expression was the highest in head kidney and weak in brain and blood. We also performed qRT-PCR on three small RNA libraries (CG, NOSG and HOSG) prepared from C. semilaevis immune tissues. The results showed that the expression of miR-223 was very diverse, depending on different tissues and samples. In response to the infection of V. anguillarum at different time-points, there was a great difference in the expression of miR-223 between the control and the challenged group, in tissues including head kidney, intestine, liver and spleen. miR-223 was up-regulated in liver, spleen, and head kidney, and down-regulated in intestine. We infected head-kidney cells with LPS, poly I:C, PGN and RGNNV. It was found that the expression of miR-223 was up-regulated in response to LPS and RGNNV, and was down-regulated in response to PGN and poly I:C. The sequence of miR-223 precursor was obtained through gene cloning and expression profile analysis after infection with bacteria or virus, and it was proved that miR-223 might play a key role in the innate immune response to pathogens. Our study shed lights on the molecular functions of miRNAs in the host-pathogen interaction in C. semilaevis.