miR-434 and miR-242 have a potential role in heat stress response in rainbow trout (Oncorhynchus mykiss).

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.