Illumina Sequencing Reveals Aberrant Expression of MicroRNAs and Their Variants in Whitefish (Coregonus lavaretus) Liver after Exposure to Microcystin-LR

Abstract

Molecular analyses show that challenging fish with microcystin-LR (MC-LR) causes perturbations of microRNA (miRNA) signaling. However, the significance and scope of these alterations is currently unknown. To address this issue, we studied miRNA gene expression in the liver of juvenile whitefish, C. lavaretus, during 28 days of exposure to a subacute dose of MC-LR (100 μg·kg-1 body mass). Using genomic resources of Atlantic salmon (AGKD03), the mature miRNA library of Atlantic salmon (miRBase-21) and bioinformatics tools (sRNAbench), we discovered and annotated a total of 377 distinct mature miRNAs belonging to 93 families of evolutionary conserved miRNAs, as well as 24 novel mature miRNA candidates that were mapped to 14 distinct S. salar miRNA precursors. miRNA-Seq transcriptome profiling of liver tissues revealed differential miRNA expression in control and treated fish at 14 days (73 miRNAs were modulated) and at 28 days (83 miRNAs) of the treatment, subsequently validated by qPCR for nine selected differentially expressed miRNAs. Additional qPCR study confirmed the miRNA-Seq data and revealed consistent, aberrant miRNAs expression profile in the later phase of MC-LR hepatotoxicity (7–28 d). Functional annotation analysis revealed that the aberrantly expressed miRNAs have target genes involved in cytoskeletal remodeling, cell metabolism, cell cycle regulation and apoptosis; dysregulation of these processes in liver cells leads to cirrhosis and hepatocellular carcinoma in humans. To enable deeper insight into the molecular responses of liver cells in fish exposed to MC-LR, we expanded the miRNAome analysis by inclusion of miRNA variants (isomiRs) profiles, and we showed that the isomiR profiles of liver specific MiR122, and a few other miRNAs, correlated with MC-LR treatment. Given the importance of isomiRs for disease biology in mammals, we believe that further research focused on the miRNA isoforms will bring us closer to better understanding the molecular mechanisms of MC-LR hepatotoxicity.

De novo profiling of long non-coding RNAs involved in MC-LR–induced liver injury in whitefish: discovery and perspectives

“…Our previous results showed that repeated exposure of white sh to MC-LR results in severe liver damage, followed by an unexpected resilience to further exposures to the toxin and regeneration of the damaged liver structure [14]. We showed that in these adaptations, MC-LR regulates several hepatic miRNA signaling pathways and alters the expression pro les of miRNAs over the short-term [15] and long-term [16], suggesting extensive transcriptome rebuilding during these processes. Because lncRNAs have been implicated in species-speci c adaptations (e.g.…”

“…The dose of MC-LR (100 μg•kg -1 of body mass) and the treatment periods (1, 6, and 9 days) were based on our previous studies on molecular and physiological responses of white sh to this toxin [14,16,21]. MC-LR (purity ≥ 95%; HPLC) was obtained from Enzo Life Sciences (Enzo Biochem, Inc.; USA) and dissolved in phosphate buffer saline (PBS) as a solvent vehicle.…”

“…Each experimental group (PBS or MC-LR) in each treatment period (1, 6 and 9 days) consisted of n = 6 individuals, thus in total 24 individuals were used in our MC-LR-treatment study. The number of sh in each experimental group were estimated based on our previous work [16], where we demonstrate that this group size is su cient to observe a distinct effect of MC-LR on ncRNA expression pattern.…”

Discovery of isomiRs in PBMCs of diseased vis-à-vis healthy Indian water buffaloes

“…The miRNA study performed on whitefish (Coregonus lavaretus) revealed that exposure to microcystin-LR results in perturbation on hepatic miRNA signaling pathway. The results concluded that miRNA and its variants can be used as markers for identification of liver-specific diseases in mammals [9]. Some miRNA and target genes, which are associated with human diseases are also well conserved in domestic animals.…”

“…The isomiRs, i.e. the sequence variants of known miR-NAs, are gaining importance in human, rat, mice and other species for their putative role in various diseases [9][10][11]. It is sometimes evident that the canonical sequence of miRNAs encoded by the same gene displays variation in length and sequence because of addition/ deletion of one or more bases at any one or both the termini [12].…”