草鱼miR-23a在嗜水气单胞菌感染过程中的调控机制

为探索嗜水气单胞菌感染后草鱼肾脏细胞(Ctenopharyngodon idella kidney,CIK)中miR-23a的调控机制,实验通过实时荧光定量PCR(qPCR)测定了嗜水气单胞菌感染CIK细胞后miR-23a的表达量变化,使用RNAhybrid软件预测miR-23a的靶基因并用双萤光素酶报告基因检测系统进...

Regulation mechanism of miR-23a in Ctenopharyngodon idella during Aeromonas hydrophila infection

Grass carp (Ctenopharyngodon idella) is the most productive aquaculture fish in the world. With the development of C. idella culture, diseases have occurred from time to time, and the disease caused by Aeromonas hydrophila infection lead to serious economic losses. miRNA is a kind of endogenous non-coding small molecule RNA with a length of about 22 nt, which can bind to 3''-UTR (3''-untranslated regions) of target mRNA to inhibit the translation of target mRNA or degrade target mRNA. As a new type of gene expression regulation molecule, miRNA can participate in the regulation of different types of biological processes such as cancer occurrence, immunity, development, cell differentiation, apoptosis and immune defense. The degree of complementarity corresponds to different forms of action. When miRNA and target mRNA 3''-UTR have incomplete complementary binding, protein production is inhibited at the translation level. When miRNA completely complements the target mRNA 3''-UTR, degradation generally occurs after transcription. At present, more than 30 000 miRNA molecules have been found in more than 200 species. In all kinds of aquatic animals, miRNAs perform a variety of biological functions in their bodies. For example, some miRNAs are closely related to the regulation of immune response in bony fish after virus or bacterial infection. During Vibrio anguillarum infection in ayu, miR-155 promotes pro-inflammatory functions and augments apoptosis of monocytes/macrophages. In addition, some studies have found that miRNAs such as miR-148, miR-214 and miR-19a can also inhibit the expression of MyD88, which can negatively regulate the inflammatory response caused by bacteria. In this study, the target genes of miR-23a were screened and determined to study its mechanism after A. hydrophila infection. In order to explore the regulatory mechanism of miR-23a in C. idella kidney (CIK) cells infected with A. hydrophila, the expression of miR-23a in CIK cells infected with A. hydrophila was determined by real-time quantitative PCR(qPCR). The target genes of miR-23a were predicted by RNAhybrid software and identified by dual-luciferase reporter assay system. Finally, the regulation of miR-23a on downstream genes was analyzed. The study found that the expression of miR-23a changed significantly at different time points of infection. In the dual-luciferase reporter assay and experiments of miR-23a agomir/antagomir transfection, the target genes were reversely regulated, and tbk1, glut1 were identified as the target genes of miR-23a. The expression of ldha, ldhba, pdha1a and pdha1b were suppressed after overexpression of miR-23a. These results showed that miR-23a was involved in the regulation of immune response in CIK cells after A. hydrophila infection. tbk1 and glut1 are the target genes of miR-23a. miR-23a can affect the expression of downstream genes by targeting GLUT1. The above results provide important ideas for the role of miR-23a in the regulation of immune response in bony fishes, and provide more theoretical basis for further understanding of miRNA-mediated multiple target genes to regulate the innate immunity of fish.