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.
Highlights
Microcystin-LR (MC-LR) is one of the most commonly occurring toxin produced by cyanobacteria of the genera Microcystis, Anabaena, Planktothrix, Chroococcus, that has received worldwide concern in recent decades [1]
MC-LR was found capable of triggering oxidative stress in liver cells which results in alterations in the cytoskeleton, leads to a loss of cell shape, cellular damage and to the liver injury [11,14]
To exclude lowly expressed inclusion of miRNA variants (isomiRs), we considered for statistical analysis, except for differential expression study, only isomiRs that had more than 100 reads in at least one sample
Summary
Microcystin-LR (MC-LR) is one of the most commonly occurring toxin produced by cyanobacteria of the genera Microcystis, Anabaena, Planktothrix, Chroococcus, that has received worldwide concern in recent decades [1]. MC-LR was found capable of triggering oxidative stress in liver cells which results in alterations in the cytoskeleton, leads to a loss of cell shape, cellular damage and to the liver injury [11,14]. Not surprisingly, these destructive processes have been contrasted by evidence that MC-LR triggers cell specific repair programs such as cell cycle arrest [13] or apoptosis both in vivo [15] and in vitro [16]. Concerns are focused mainly on identification of particular hallmarks of the aberrant processes leading to MC-LR induced-liver toxicity and disease (e.g. [17,18])
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