Identification and characterization of miRNA involved in white spot syndrome virus infection in black tiger shrimp Penaeus monodon

Abstract

MicroRNA (miRNA) is a small RNA that functions in regulating various biological processes including immune system by suppressing the gene expression via either mRNA degradation or inhibiting gene translation. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon hemocyte were identified by next generation sequencing technique. Three small RNA libraries of hemocyte of WSSV-infected P. monodon at 0, 6 and 48 hours post infection (hpi) were constructed using TruSeq small RNA sample preparation kits and subjected to MiSeq sequencer (Illumina). Then, the raw data whose quality could pass the quality filter was processed by removing 3’- and 5’-adapter sequences and known contaminated-RNAs. The remaining sequences were searched against miRNA database, miRBase, to identify miRNA homologs. In this study, sixty miRNA homologs from WSSV-infected P. monondon hemocyte were identified. In order to analyze the expression of interested miRNAs in response to WSSV challenge, Northern blot analysis of 6 interested miRNAs was performed. Only 2 of them, let-7-5p and miR-71-5p could be detected and they were differentially expressed in hemocyte of WSSV-challenged P. monodon. Due to the detection limit of probes used in Northern blot analysis, stem-loop real-time PCR was used instead to further determine the expression of 16 interested miRNAs in hemocyte of WSSV-challenge P. monodon at 0, 6 and 48 hpi. The results showed that 11 out of 16 miRNAs were differently expressed upon WSSV infection. Two miRNAs, miR-315 and miR-750, were highly responsive miRNAs upon WSSV infection. To further characterize the involvement of the identified miRNAs in P. monodon immunity against WSSV infection, target mRNAs of each miRNA were predicted by in-house software against P. monodon EST database. In this study we focused on the target mRNAs that are immune-related genes of apoptosis, antimicrobial peptides, prophenoloxidase system, proteinase and proteinase inhibitor, signaling transduction and heat-shock protein. From the prediction, 46 out of 60 identified P. monodon miRNAs were targeted at 5’UTR, ORF and 3’UTR regions of several immune-related genes. These results implied that these miRNAs might play roles as immune gene regulators in shrimp antiviral response.