| Fish,which are ectothermic(heterothermic)animals.The temperature of the environment is a critical factor for fish growth,development,immune function,metabolism and breeding of fish.In particular,a rapid drop in temperature poses a major challenge to the survival of poikilothermic fish,and in severe cases can even lead to their death,which includes aquaculture species with important commercial value.Therefore,it is of great value and significance to study fish under cold adaptation and cold stimulation.Zebrafish(Danio rerio),a fresh water tropical cyprinid fish with short breed cycle and big spawn amount,is one of the most important vertebrate models in genetics,development,and biomedicine.Zebrafish is also a good model to study cold acclimation with a wide temperature range of approximately 16?C-40?C.Our previous research showed that DNA methylation and long non-coding RNAs(lnc RNAs)are involved in cold acclimation of zebrafish ZF4 cells when experimentally acclimated at 18?C for 30 d.MicroRNAs(miRNAs)are a class of single-stranded endogenous non-coding RNAs in plants and animals,approximately 22 nucleotides in length.As post-transcriptional regulators of gene,miRNA combine with 3’UTR of mRNAs with perfect or near perfect complementarities by complementary base pairing,leading to mRNA cleavage and translational repression of their corresponding target protein-coding genes.A wide range of studies have shown that miRNAs are involved in the regulation of plant and animal adaptation to low temperature pressures.However,current studies on hypothermia stress mainly focus on plants,and there are also some studies on mammalian responses to acute hypothermia stress.Studies on fish mainly focus on genome and transcriptome,while studies on relationship between fish hypothermia adaptation and miRNA regulation are rare.To identify miRNAs in response to low temperature stress in zebrafish in the present study,high-throughput sequencing was performed to identify differentially expressed miRNAs between the zebrafish embryonic fibroblast ZF4 cells under 28°C,30 d(control)and 18°C,30 d(cold-acclimation)conditions.The results are as follows:1.The zebrafish embryonic fibroblast ZF4 cells with a normal culture temperature of 28°C were transferred to an incubator at 18?C for 30 days as cold acclimation group.The ZF4 cells under normal culture conditions(28°C)were cultured for 30 days as control group.High-throughput sequencing was performed to identify differentially expressed miRNAs between two groups.A total of 414 miRNAs,349 known and 65 novels were identified through bioinformatics analysis.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 by DEseq.2.Target prediction of DE-miRNAs(differentially expressed miRNA)were performed using the miRanda software to explore the role of miRNAs in cold acclimation of zebrafish ZF4 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,ECMreceptor interaction and so on.3.Moreover,both miR-100-3p inhibitor and miR-16 b mimics could protect ZF4 cells under the cold stress of 10°C,indicating the key functions of miRNA in cold acclimation.Further study showed that miR-100-3p and miR-16 b could repress the expression of their target gene(atad5a,cyp2ae1,lamp1,rilp,atxn7,tnika,btbd9)4.The role of miR-100-3p in zebrafish embryonic development was further investigated by microinjection of miR-100-3p mimics or inhibitor.From 6 hpf(hours post fertilization)miR-100-3p mimics injected embryos showed an increased mortality rate,and no significant change was observed in embryonic mortality in miR-100-3p inhibitor injected embryos.Our results suggested that miR-100-3p overexpression affects the early development of zebrafish embryos.In this study,we systematically characterized the variation of miRNAs in zebrafish embryonic fibroblast ZF4 cells under cold acclimation conditions through highthroughput sequencing.There are 28 conserved and 19 novel miRNAs differentially expressed in cold acclimation in ZF4 cells.Those DE-miRNAs are involved in regulation of phosphorylation,cell junction,intracellular signal transduction,ECM-receptor interaction and so on.Further study showed that dre-miR-16 b and dre-miR-100-3p contributed to cold acclimation through regulating cell survival.In conclusion,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. |
