Identification and characterization of miRNAs involved in cold acclimation of zebrafish ZF4 cells

Abstract

MicroRNAs (miRNAs) play vital roles in various biological processes under multiple stress conditions by leading to mRNA cleavage or translational repression. However, the detailed roles of miRNAs in cold acclimation in fish are still unclear. In the present study, high-throughput sequencing was performed to identify miRNAs from 6 small RNA libraries from the zebrafish embryonic fibroblast ZF4 cells under control (28°C, 30 days) and cold-acclimation (18°C, 30 days) conditions. A total of 414 miRNAs, 349 known and 65 novel, were identified. Among those miRNAs, 24 (19 known and 5 novel) were up-regulated, and 23 (9 known and 14 novel) were down-regulated in cold acclimated cells. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses indicated that the target genes of known differentially expressed miRNAs (DE-miRNA) are involved in cold acclimation by regulation of phosphorylation, cell junction, intracellular signal transduction, ECM-receptor interaction and so on. Moreover, both miR-100-3p inhibitor and miR-16b mimics could protect ZF4 cells under cold stress, indicating the involvement of miRNA in cold acclimation. Further study showed that miR-100-3p and miR-16b could regulate inversely the expression of their target gene (atad5a, cyp2ae1, lamp1, rilp, atxn7, tnika, btbd9), and that overexpression of miR-100-3p disturbed the early embryonic development of zebrafish. In summary, the present data show that miRNAs are closely involved in cold acclimation in zebrafish ZF4 cells and provide information for further understanding of the roles of miRNAs in cold acclimation in fish.

Fig 1. Pipeline workflow of small RNA search and target prediction.

miRBase: microRNAs database, Ref ncRNA: reference non-coding RNA sequences, miRNAs: microRNAs, sRNAs: small RNAs, DE-miRNAs: differentially expressed miRNAs, DAVID: Database for Annotation, Visualization and Integrated Discovery, GO: Gene Ontology, KEGG: Kyoto Encyclopaedia of Genes and Genomes.

Fig 2. Statistics and classification of small RNAs.

Read length distribution of small RNA libraries (A) and nucleotides bias at each position of miRNA (B) for control or cold acclimation condition. (C) An overview of the frequency of different RNA species present in libraries from each group. miRNA: microRNA, unmatched: unmatched reads, rRNA: ribosomal RNA, lincRNA: long intergenic non-coding RNA, snoRNA: small nucleolar RNA, snRNA: small nuclear RNA, misc_RNA: miscellaneous RNA, Mt_tRNA: mitochondrial transfer RNA, Mt_rRNA: mitochondrial ribosomal RNA, scaRNA: small cajal body-specific RNA.

Fig 3. Identification and annotation of small RNAs.

(A) Comparison of miRNA expression levels between small RNA libraries from control and cold acclimated ZF4 cells. (B) Venn diagram shows the number of known miRNAs expressed only in control and cold acclimated ZF4 cells. (C) A heatmap was generated based on fold change values of known DE-miRNAs to visualize the expression patterns of the cold responsive miRNAs. (D) ZF4 cells were cultured at 18°C for 30 days, then returned to 28°C and cultured for another 10 days for recovery. The expression of indicated miRNAs was detected using qRT-PCR. The data are presented as means ± SD of three independent replicates. p <0.05 was considered to indicate a statistically significant result. *: p <0.05, **: p <0.01, ***: p <0.001.

Fig 4. Identification and functional prediction of DE-miRNAs.

(A-B) GO and KEGG enrichment analyses of the target genes of DE miRNAs. The y-axis corresponds to GO/ KEGG pathway with a p-value ≤ 0.05, and the x-axis shows the enrichment ratio between the number of DE-genes and all unigenes enriched in a particular pathway. The color of the dot represents p-value, and the size of the dot represents the number of DE-genes mapped to the reference pathways.

Fig 5. Effect of selected miRNAs on their target genes.

ZF4 cells were transfected with indicated mimics or inhibitor, 24 hours later the expression of miR-100-3p or miR-16b (A-B) and their target genes (C-F) was detected by qRT-PCR. NC: negative control. *: p <0.05, **: p <0.01, ***: p <0.001.

Fig 6. dre-miR-100-3p and dre-miR-16b affect viability of ZF4 cells and embryonic development of zebrafish.

(A-B) ZF4 cells were transfected with indicated mimics or inhibitor, 24 hours later the cells were exposed to 10°C for 36 hours, then cell viability was examined. (C-D) Zebrafish embryos were microinjected with indicated mimics or inhibitor, 24 hours later the expression of miR-100-3p was detected by qRT-PCR (C), and viability of embryos was determined (n = 120) (D). *: p <0.05, **: p <0.01, ***: p <0.001. NC, negative control.