Search

MicroRNAs (miRNAs) are being extensively studied as they function as key metabolic regulators which play a role in the heat stress response. However, the role of miRNAs in heat stress remains uncertain and many new miRNAs have not yet been discovered. In a previous study, we performed high‐throughput sequencing of differentially expressed miRNAs identified on exposing rainbow trout (Oncorhynchus mykiss) to heat stress (18 vs. 24°C), which led to the identification of two novel miRNAs, temporarily named novel miR‐434 and ‐242. The differential expression level of these miRNAs was extremely significant (P < 0.01); we analysed target gene mRNA transcripts by bioinformatics software (miRanda). We found novel miR‐434 and ‐242 were predicted to regulate the transcripts of heat shock 70‐kDa protein 4‐like (HSPA4L) and calreticulin (CRT), respectively, by bioinformatics software. Here our core objective was to validate if HSPA4L and CRT are indeed the target genes of novel miR‐434 and ‐242, respectively, and for this purpose we used the dual‐luciferase reporter assay system. Target gene sequences were synthesized and cloned into a dual‐luciferase vector. To better understand the function of the target genes, we combined the previous sequencing results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. We found that novel miR‐434 regulated HSPA4L expression by binding to a putative binding site in the 3′‐UTR of HSPA4L, and luciferase activity inhibition was observed. In contrast, novel miR‐242 was not involved in regulating CRT expression. To conclude, we believe our results should serve as a foundation for future studies aiming to comprehensively understand the mechanisms used by rainbow trout to cope with heat stress. [ABSTRACT FROM AUTHOR]

Abstract MicroRNAs (miRNAs) play key roles in the stress response in animals. The rainbow trout is a widely cultivated cold-water fish species that usually encounters heat stress, which affects its growth and may cause diseases. However, there is limited information on the miRNAs involved in heat stress in this species. To identify the heat-responsive miRNAs at the genome level in rainbow trout, we conducted high-throughput sequencing of 6 small RNA libraries from the liver tissues of rainbow trout specimens under control and heat-treated conditions. A total of 363 conserved miRNAs and 638 novel miRNAs were identified, among which 39 (21 conserved and 18 novel) miRNAs were differentially expressed in response to heat stress. Eight important miRNAs were further validated using quantitative real-time PCR. GO enrichment and KEGG pathway analyses of the potential target genes of the DE miRNAs, revealed DNA replication and protein folding and unfolding were affected by heat stress. Several key pathways involved in the heat stress response were characterized, such as protein processing in the endoplasmic reticulum, NOD-like receptor signaling pathway, and progesterone-mediated oocyte maturation. In addition, 20 important functional interaction pairs among the 65 negatively correlated miRNA-mRNA pairs were identified using integrated miRNA-mRNA analysis and miRNA prediction algorithms, which including potential important miRNAs and target genes such as miR-301a, miR-301d, let-7c, novel_1181 and HSP90BA , HSP90BB , HSP40 , PDIA3. This study showed that the identified miRNAs probably play important roles in the heat stress response and lay a foundation for further studies on the thermal adaptation mechanisms in rainbow trout. Highlights • We sequenced 6 miRNA libraries from rainbow trout livers in 18 °C and 24 °C. • A total of 363 conserved miRNAs and 638 novel miRNAs were identified. • 21 conserved and 18 novel miRNAs were differentially expressed. • 20 important miRNA-mRNA interaction pairs were involved in heat stress response. [ABSTRACT FROM AUTHOR]