Abstract: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.
