In vivo screening of zebrafish microRNA responses to bacterial infection and their possible roles in regulating immune response genes after lipopolysaccharide stimulation

Micro (mi)RNAs are abundant small noncoding RNAs found in plants and animals, the regulatory functions of which are not fully understood in fish. To identify potential miRNAs, we screened an miRNA microarray with total RNA from zebrafish infected with Vibrio harveyi and another from uninfected zebrafish. Six miRNAs were obtained from the microarray screening. We studied miRNA expression patterns of 2 miRNAs (miR-122 and miR-194) after bacterial infection of transgenic zebrafish (containing tilapia hepcidin (TH)2-3) and non-transgenic zebrafish from which the 2 miRNAs were obtained from the microarray experiment. The results indicated that miR-122 and miR-194 were higher in PBS-injected zebrafish compared with TH2-3 zebrafish or wild-type (WT) zebrafish after V. harveyi infection. Overexpression of miRNAs (miR-122, miR-192, and miR-194a) was seen in zebrafish liver (ZFL) cells after lipopolysaccharide (LPS) treatment and in untreated fish. Our results showed that after 24 h of doxycycline treatment without LPS stimulation, interleukin (IL)-22, lysozyme, toll-like receptor (TLR)1, TLR3, TLR4a, and tumor necrosis factor (TNF)-alpha gene expressions were, respectively, upregulated by 14-, 22-, 2.2-, 13-, 200-, and 38-fold in miR-122-transfected compared with non-transfected (WT) ZFL cells. In cells transfected with miR-192 and treated with LPS after 8-12 h, IL-22, lysozyme, TLR1, TLR3, TLR4a, and TNF-alpha expressions significantly differed between WT and miR-192-overexpressing ZFL cells. However, we observed significantly higher IL-22 expression levels after 12 h of LPS treatment in miR-192-transfected ZFL cells compared with non-transfected cells. In contrast, IL-22, lysozyme, and TNF-alpha were markedly upregulated (> 100-fold) after miR-194a transfection and overexpression in ZFL cells and treatment with LPS. Our cloning and expression analyses indicated that miR-122, miR-192, and miR-194a play important roles in zebrafish immunology.