In vivo screening of zebrafish microRNA responses to bacterial infection and their possible roles in regulating immune response genes after lipopolysaccharide stimulation
In vivo screening of zebrafish microRNA responses to bacterial infection and their possible roles in regulating immune response genes after lipopolysaccharide stimulation
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DOI：
10.1007/s10695-012-9617-1
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发表时间：
2012-10-01
影响因子：
2.9
通讯作者：
Chen, Jyh-Yih
中科院分区：
文献类型：
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作者：
Wu, Tsung-Han;Pan, Chieh-Yu;Chen, Jyh-Yih
微RNA是在动植物中发现的大量非编码小RNA，其在鱼类中的调控功能尚不完全清楚。为了鉴定潜在的miRNA，我们用感染哈氏弧菌的斑马鱼的总RNA和另一种未感染的斑马鱼的总RNA筛选了一个miRNA微阵列。从微阵列筛选中获得6个miRNAs。我们研究了细菌感染转基因斑马鱼（含有罗非鱼hepcidin（TH）2-3）和非转基因斑马鱼后2种miRNA（miR-122和miR-194）的miRNA表达模式，从微阵列实验中获得了2种miRNA。结果表明，在哈维氏弧菌感染后，PBS注射的斑马鱼中的miR-122和miR-194高于TH 2 -3斑马鱼或野生型（WT）斑马鱼。在脂多糖（LPS）处理后的斑马鱼肝（ZFL）细胞和未处理的鱼中观察到miRNA（miR-122、miR-192和miR-194 a）的过表达。我们的结果显示，在没有LPS刺激的情况下，强力霉素处理24小时后，白细胞介素（IL）-22，溶菌酶，toll样受体（TLR）1，TLR 3，TLR 4a和肿瘤坏死因子（TNF）-α基因表达分别上调14-，22-，2.2-，13-，200-，与未转染（WT）ZFL细胞相比，在miR-122转染的ZFL细胞中是38倍。在用miR-192转染并用LPS处理8-12 h后的细胞中，IL-22、溶菌酶、TLR 1、TLR 3、TLR 4a和TNF-α表达在WT和miR-192过表达的ZFL细胞之间显著不同。然而，与未转染的细胞相比，我们在miR-192转染的ZFL细胞中观察到LPS处理12小时后IL-22表达水平显著更高。相反，在ZFL细胞中miR-194 a转染和过表达以及用LPS处理后，IL-22、溶菌酶和TNF-α显著上调（> 100倍）。我们的克隆和表达分析表明miR-122、miR-192和miR-194 a在斑马鱼免疫学中起重要作用。
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.