Regulation Of Non-coding RNA Response To Heat Stress And Its Functional Verification In CeRNA Mechanism In Rainbow Trout(Oncorhynchus Mykiss)

Rainbow trout(Oncorhynchus mykiss),belonging to Salmon genus,Salmonidae,is a typical economically important cold-water fish.However,rainbow trout has poor tolerance to higher temperature,the optimal growth temperature for rainbow trout are 12 ? to 18 ?,and must be breeding under flowing water or micro-flowing water conditions.Heat stress has become one of the major environmental problems faced by aquaculture industry in the context of global warming and frequent extreme high temperature weather.Therefore,in China,high temperature in summer is the biggest threat in rainbow trout breeding,which is easy to cause the reduction of rainbow trout production or the death of a large area.The harm of heat stress on rainbow trout has attracted more and more scholars' attention and research.In recent years,the important regulatory roles of non-coding RNAs in the processes of growth,development and stress of the organism have attracted widespread attention.The regulation of target genes(m RNA)mediated by non-coding RNAs(such as mi RNA and lncRNAs)may be the main strategy for enhancing adaptability in response to heat stimulation.Therefore,in order to further reveal the regulatory mechanism of noncoding RNA in rainbow trout liver response to heat stress,this study studied on the molecular mechanism of non-coding RNA and its target genes in the regulation of endogenous competitive RNA(ceRNA)in the liver tissues of the normal group(18?)and the heat stress group(24?)through high-throughput sequencing and bioinformatics analysis.And the targeting relationship between the key novel-m0007-5p and hsp90ab1,LOC110485411 was further verified by dual luciferase reporter gene assay.The role of LOC110485411,novel-m0007-5p and hsp90ab1 in the regulation of heat stress was further verified at the cellular level by establishing the primary hepatocyte model of rainbow trout.The aim is to reveal the molecular regulation mechanism of non-coding RNA in response to heat stress,so as to provide scientific basis for the formulation of drug research and development strategies for alleviating heat stress of rainbow trout and improvement of heatresistant strains.The main results of this study were as follows:1? In this study,a total of 4138 circRNAs,5916 lncRNAs,67107 m RNAs and 2730 mi RNAs were obtained from 6 libraries(CG=3,HS=3)through the whole transcriptome sequencing of rainbow trout liver tissues under chronic heat stress.A total of 324 circRNAs,428 lncRNAs,105 mi RNAs and 1885 m RNAs with significant differences in expression were screened by strict thresholds.The differentially expressed non-coding RNAs indicated that they played an important role in the response of rainbow trout liver to heat stress.2? The ceRNA regulatory network of circRNA-mi RNA-m RNA was constructed,and some important regulatory relationships were predicted,such as novel?circ?003889-novelm0674-3p-hsp90ab1,novel?circ?002325-mi R-18-y-HSPA13 and novel?circ?002446-novel-m0556-3p-hsp70.Some genes involved in the biological regulation of metabolic processes or in response to stimulation were highly induced under heat stress.Some important pathways related to heat stress,such as ER protein processing,estrogen signaling pathway and HIF-1 signaling pathway were significantly enriched.This suggests that circRNA may affect HSPS protein expression in rainbow trout liver response to heat stimulation through regulation of mi RNA,and these effects may be related to protein folding,processing and other pathways.3? The ceRNA regulatory network of lncRNA-mi RNA-m RNA was constructed,and some important regulatory pathways,such as endoplasmic reticulum protein processing pathway,antigen processing pathway and estrogen signaling pathway,NOD-like receptor signaling pathway,MAPK signaling pathway,PI3K-Akt signaling pathway,were significantly enriched.Two important regulatory pairs were found: LOC110485411-novelm0007-5p-hsp90ab1 and LOC110506561-mi R-8159-x-hsp90 a.1.This suggests that lncRNAs can affect the expression of target gene proteins by regulating the expression of novel-m0007-5p,mi R-8159-x and other mi RNAs in the process of rainbow trout liver response to heat stimulation,so as to exert the anti-heat stress function of lncRNAs.4? The targeting relationship between LOC110485411,novel-m0007-5p and hsp90ab1 was predicted by bioinformatics method,and the wild-type recombinant vector(pmir GLO-hsp90ab1-WT and pmir GLO-LOC110485411-WT)and the mutant recombinant vector(pmir GLO-hsp90ab1-MUT and pmir GLO-LOC110485411-MUT)were constructed,and the targeting relationship between LOC110485411 & novel-m0007-5p and hsp90ab1 &novel-m0007-5p was verified by dual luciferase reporter gene assay,respectively.This suggests that LOC110485411 and hsp90ab1 are target genes of novel-m0007-5p,and we speculate that LOC110485411 may exert regulatory effects through novel-m0007-5P.5? Exogenous novel-m0007-5p mimics and inhibitor were transfected into primary hepatocytes of rainbow trout,and it was found that they could effectively bind to target genes hsp90ab1 and LOC110485411 to exert inhibitory or promoting effects,and had no significant effects on the proliferation and apoptosis of hepatocyte viability.Overexpression of novel-m0007-5p under heat stress showed time-sensitive inhibition of target genes hsp90ab1 and LOC110485411.Similarly,si RNA silencing the expression of LOC110485411 is also time-sensitive,and si RNA can affect the expression of hsp90ab1 m RNA by silencing the expression of LOC110485411.This suggests that there is a regulatory mechanism of ceRNA in the response of primary rainbow trout hepatocytes to heat stimulation.LOC110485411 affects the expression of target gene hsp90ab1 by regulating the expression of novel-m0007-5p,so as to resist heat stimulation.