Effects Of MiR-183-5p On Zebrafish Cells Under Low Temperature Pressure

MicroRNAs(miRNAs)are approximately 21 nucleotides in length.The post-transcriptional repression through miRNA seed region binding to 3’ UTR of target m RNA is considered as the canonical mode of miRNA-mediated gene regulation.Studies have shown that miRNA,as an important regulatory factor,is involved in a variety of key biological processes in fish.Fish can change the expression of their target genes by changing the expression level of miRNA,so as to respond to changes in external pressure.A large number of studies have shown that some miRNAs can be expressed in fish under low temperature stress,and used as target genes related to low temperature resistance,so that fish can produce adaptability to low temperature in physiological response.A study analyzed transcriptional responses of turbot under cold conditions by RNA-seq and micro RNA(miRNA-seq),and found that the expression of most miRNAs tended to be up-regulated under cold conditions.The network of miRNA-transcription factor-m RNA,regulating turbot different response to cold stress,was constructed,which involved in cell cycle,component of cell membrane,signal transduction,and circadian rhythm pathways.Another study analyzed miRNA expression in zebrafish ZF4 cells after cold acclimation through miRNA-seq,and the results showed that 18 miRNAs were up-regulated and 10 miRNAs were down-regulated after cold acclimation in ZF4 cells.In order to study the effect of miR-183-5p in the low temperature domestication of zebrafish cells,the expression levels of miRNAs in zebrafish ZF4 cells after 30 days of 18°C low temperature domestication and ZF4 cells cultured at 28°C were detected by RT-q PCR.The expression levels of miR-183-5p in ZF4 cells after 30 days of low temperature domestication were significantly up-regulated,indicating that miR-183-5p is involved in the low temperature regulation of ZF4 cells.Evolutionary tree sequence analysis shows that the zebrafish precursor miR-183 is high homology of many species of miR-183-5p mature body find zebrafish multiple sequence alignment,and the miR-183-5p sequence is very conservative,that miR-183-5p in highly genetic between different species.This suggest that miR-183-5p may have the similar function in zebrafish and other species.Cell viability test verified that overexpression of miR-183-5p could enhance the viability of ZF4 cells under low temperature,indicating that miR-183-5p significantly up-regulated zebrafish cells under low temperature after30 days of low temperature domestication played a protective role in zebrafish cells.After bioinformatics predicted the target genes of miR-183-5p,it was found that egr1,fbxl7 and MPB might be the target genes of miR-183-5p.RT-q PCR confirmed that the expression of EGR1 in zebrafish ZF4 cells was significantly decreased after 5,10 and30 days of hypothermia treatment,and the expression of EGR1 in zebrafish ZF4 cells after 30 days of hypothermia treatment was significantly lower than that after 5 and 10 days of hypothermia treatment,indicating that ZF4 cells are more inclined to inhibit the expression of EGR1 in the process of hypothermia accumulation,so that the cells can adapt to the hypothermia environment.In addition,with the up regulation of mir-183-5p at low temperature,the down regulation of EGR1 was negatively regulated,which was consistent with the prediction of miR-183-5p target gene.In order to investigate the targeting effect of miR-183-5p on egr1,pmiRGLO-egr1-3’UTR recombinant plasmid was constructed and co-transfected with miR-183-5p mimics/miR-NC into HEK-293 T cells.The dual luciferase activity assay results showed that the overexpression of miR-183-5p significantly inhibited the luciferase activity expressed by the dual luciferase reporter gene vector,suggesting that miR-183-5p could directly act on the egr1 3’UTR.The phylogenetic tree and homology analysis of EGR1 showed that EGR1 was conserved among multiple species.The functional network map of EGR1 was obtained by Spike database analysis.The map showed that EGR1 was associated with Na B2 and TP53,suggesting that the interaction between EGR1 and Na B2 and TP53 could be studied in the future studies of miR-183-5p/ egr1 signaling pathway at low temperatures.This study revealed that the increased expression of miR-183-5p in ZF4 cells can enhance the viability of ZF4 cells in zebrafish under low temperature.It was verified that miR-183-5p could regulate egr1 3’UTR.Proteins interacting with EGR1 were predicted by functional network analysis of EGR1.This study confirmed that overexpression of miR-183-5p had a protective effect on cells at low temperature.The miR-183-5p/egr1 signaling pathway was verified.The interaction of EGR1 with Na B2 and TP53 was predicted.This study improved the signaling pathway of zebrafish cells under low temperature,and provided a reference for the study of the low temperature adaptation mechanism of zebrafish.