Abstract
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)-α 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-α 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-α 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.
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Acknowledgments
This work was supported by a grant from the Development Program of Industrialization for Agricultural Biotechnology to Dr. Cho-Fat Hui under the grant name, “Characterization of microRNAs in fish during normal or disease states.”
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Tsung-Han Wu, Chieh-Yu Pan and Ming-Ching Lin contributed equally to this work.
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10695_2012_9617_MOESM1_ESM.pdf
Supplementary figure 1. Detection of lipopolysaccharide (LPS)-responsive micro (mi)RNAs. In order to find miRNAs after bacterial infection, microarray spot signal intensities were subjected to a paired t-test analysis. miRNA expression patterns after a Vibrio harveyi (204) infection were compared to those of wild-type (WT-control) zebrafish. Results indicated that 6 miRNAs showed differential expression patterns. Supplementary material 1 (PDF 134 kb)
10695_2012_9617_MOESM2_ESM.pdf
Supplementary figure 2. Expression of micro (mi)RNA in cells from the transient transfection of a zebrafish liver (ZFL) cell line expressing each miRNA. Cells were collected after 24 h and treated with doxycycline (1 mg/ml), and its expression was detected using a qPCR. ZFL represents ZFL cells without transfection of the miRNA plasmid or doxycycline (1 mg/ml) treatment. TFZ + Dox represents ZFL cells transfected with the miRNA plasmid and with doxycycline (1 mg/ml) treatment. TFZ-Dox represents ZFL cells transfected with the miRNA plasmid but without doxycycline (1 mg/ml) treatment. Supplementary material 2 (PDF 136 kb)
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Wu, TH., Pan, CY., Lin, MC. et al. In vivo screening of zebrafish microRNA responses to bacterial infection and their possible roles in regulating immune response genes after lipopolysaccharide stimulation. Fish Physiol Biochem 38, 1299–1310 (2012). https://doi.org/10.1007/s10695-012-9617-1
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DOI: https://doi.org/10.1007/s10695-012-9617-1