Biological Activity and Stability of Aeruginosamides from Cyanobacteria.

Aeruginosamides (AEGs) are classified as cyanobactins, ribosomally synthesized peptides with post-translational modifications. They have been identified in cyanobacteria of genera Microcystis, Oscillatoria, and Limnoraphis. In this work, the new data on the in vitro activities of three AEG variants, AEG A, AEG625 and AEG657, and their interactions with metabolic enzymes are reported. Two aeruginosamides, AEG625 and AEG657, decreased the viability of human breast cancer cell line T47D, but neither of the peptides was active against human liver cancer cell line Huh7. AEGs also did not change the expression of MIR92b-3p, but for AEG625, the induction of oxidative stress was observed. In the presence of a liver S9 fraction containing microsomal and cytosolic enzymes, AEG625 and AEG657 showed high stability. In the same assays, quick removal of AEG A was recorded. The peptides had mild activity against three cytochrome P450 enzymes, CYP2C9, CYP2D6 and CYP3A4, but only at the highest concentration used in the study (60 µM). The properties of AEGs, i.e., cytotoxic activity and in vitro interactions with important metabolic enzymes, form a good basis for further studies on their pharmacological potential.

De Novo Profiling of Long Non-Coding RNAs Involved in MC-LR-Induced Liver Injury in Whitefish: Discovery and Perspectives.

Microcystin-LR (MC-LR) is a potent hepatotoxin for which a substantial gap in knowledge persists regarding the underlying molecular mechanisms of liver toxicity and injury. Although long non-coding RNAs (lncRNAs) have been extensively studied in model organisms, our knowledge concerning the role of lncRNAs in liver injury is limited. Given that lncRNAs show low levels of sequence conservation, their role becomes even more unclear in non-model organisms without an annotated genome, like whitefish (Coregonus lavaretus). The objective of this study was to discover and profile aberrantly expressed polyadenylated lncRNAs that are involved in MC-LR-induced liver injury in whitefish. Using RNA sequencing (RNA-Seq) data, we de novo assembled a high-quality whitefish liver transcriptome. This enabled us to find 94 differentially expressed (DE) putative evolutionary conserved lncRNAs, such as MALAT1, HOTTIP, HOTAIR or HULC, and 4429 DE putative novel whitefish lncRNAs, which differed from annotated protein-coding transcripts (PCTs) in terms of minimum free energy, guanine-cytosine (GC) base-pair content and length. Additionally, we identified DE non-coding transcripts that might be 3' autonomous untranslated regions (3'UTRs) of mRNAs. We found both evolutionary conserved lncRNAs as well as novel whitefish lncRNAs that could serve as biomarkers of liver injury.

Luciferase reporter assay for small-molecule inhibitors of MIR92b-3p function: Screening cyanopeptolins produced by Nostoc from the Baltic Sea.

We developed a cell sensor that detects the liver cancer-specific microRNA MIR92b-3p, involved in hepatocellular carcinoma development and hepatitis C virus infection. To validate our small-molecule screen that employs a Huh7 human hepatoma cell line stably transfected with a pmirGLO vector containing dual luciferase reporters, we used i) a MIR92b-3p antisense or a MIR92b-3p mimicking agent (concentrations from 0.1 pM to 100 nM), ii) expression of XIST, a long non-coding RNA that is a cellular target of MIR92b, and iii) ectopic expression of Luc2 luciferase. This reporter system was used to test four cyanopeptolins from a de novo library of peptides that were isolated from the Baltic Sea cyanobacteria Nostoc edaphicum strain CCNP1411. Exposure of the Huh7-pmirGLO-MIR92b-3p cells to increasing concentrations (from 10 nM to 100 µM) of the cyanopeptolins and microcystin-LR (MC-LR; a treatment control) did not lead to a dose-dependent restoration of the luciferase signal. Instead, when the reporter cells were treated with MC-LR, the luciferase signal decreased markedly, most likely due to non-target, toxic effects of MC-LR on the cells. Although the first use of this reporter system to screen selected Nostoc peptides did not identify inhibitors of MIR92b, this method provides a means to identify functional miRNA regulators and could be readily extended to other compounds.

Molecular characterization of the cyclin-dependent protein kinase 6 in whitefish (Coregonus lavaretus) and its potential interplay with miR-34a.

Cyclin-dependent protein kinase 6 (CDK6) plays a pivotal role in the regulation of the cell cycle and cell proliferation in mammals, and disruption of its expression by various microRNAs has been implicated in the pathogenesis of multiple human cancers. In mammals, miR-34a acts as a downstream effector of p53, and thus indirectly targets Cdk6, abrogating its effects. However, no studies have been done so far to examine the mechanistic involvement of miR-34a in the silencing of cdk6 in fish. In the present study, we found that the cDNA sequence of whitefish cdk6 has a 3'UTR region that contains a binding site for miR-34a. Using a luciferase reporter assay, we demonstrated that whitefish cdk6 is a direct target of miR-34a in vitro. In order to confirm this relationship in vivo, we measured the miR-34a and cdk6 mRNA expression patterns in the liver of whitefish after short-term (8, 24, and 48 h) and long-term (14 and 28 days) exposure to microcystin-LR (MC-LR), a known hepatotoxin and tumor promoter. In contrast to the in vitro findings, we noticed an up-regulation of miR-34a and cdk6 expression after long-term MC-LR treatment. While these results indicate that both, miR-34a and cdk6 are responsive to MC-LR treatment, they do not support the presence of a miR-34a:cdk6 mRNA regulatory pair in the MC-LR-challanged whitefish liver in vivo. On the other hand, our findings suggests that cell regulatory elements, partnering with either miR-34a or cdk6, are worthy of further screening to better understand the molecular mechanisms that underlie the physiological response of fish challenged with hepatotoxic environmental pollutants like microcystins.

In vivo miRNA delivery in whitefish: Synthetic MiR92b-3p uptake and the efficacy of gene expression silencing.

IMPACT STATEMENT: The delivery of short snippets of RNA, such as synthetic miRNA agents, is an essential step for achieving RNA-mediated knockdown, which has not been studied in sufficient detail in fish. Our results indicate that a MiR92b-3p mimic may be effectively delivered via intraperitoneal injection to the spleen and the liver of whitefish, and that it likely achieves functionality without causing any apparent toxic effects in the challenged animals. We report the novel finding that the MiR92b-3p mimic reduced the in vivo liver mRNA expression levels of its putative pro-apoptotic targets (p53, cdkn1a, and pcna), and important metabolic genes, e.g. cdo1. This shows that this methodology of MiR92b-3p mimic transfection in vivo may be a useful tool for studies that investigate the molecular pathways that confer pro-proliferative and anti-apoptotic phenotypes or those that regulate intracellular metabolism in fish and other vertebrates.

Microcystin-LR-Triggered Neuronal Toxicity in Whitefish Does Not Involve MiR124-3p.

Microcystin-LR (MC-LR) is a potent hepatotoxin that has also been pointed out of causing neurotoxicity, but the exact mechanisms of action still remain ambiguous and need to be elucidated. Data from studies on mammals show that pathology of astrocyte cells points to perturbations of microRNA signaling. Glial fibrillary acidic protein (GFAP), a neuronal cell/astrocyte-specific protein, and a microRNA-124-3p (MiR124-3p) are among putative triggers and regulators of neuronal cell/astrocyte reactivity. In the present study on whitefish (Coregonus lavaretus), we found that gfap mRNA contains a putative target site for MIR124-3p, to potentially affect its expression changes. qPCR expression study of gfap:MiR124-3p pair in the midbrain of juvenile whitefish, during 28 days of exposure to a repeated subacute dose of MC-LR (100 µg kg-1 body mass), showed marginally significant up-regulation of gfap only on the 7th day of exposure period which suggests neuronal toxicity. During the whole exposure period, neither midbrain nor blood plasma levels of MiR124-3p were changed. Furthermore, double luciferase gene reporter assay confirmed the lack of MiR124-3p involvement in mediating control over gfap mRNA expression. These data show that, although MC-LR may trigger neuronal toxicity in whitefish, this does not involve MiR124-3p in response to the treatment.

Feed contamination with zearalenone promotes growth but affects the immune system of rainbow trout.

To investigate the effects of feed contamination with zearalenone (ZEN) at the current European Commission (EC) guidance value (2 mg⋅kg-1 feed) on the growth and health of rainbow trout, we performed a long-term feeding trial under aquaculture conditions. It started with the external feeding of the fish larvae, and continued for 96 weeks, at which point the fish had reached market size. To assess the growth of fish and their feeding efficiency throughout this period, the fish were regularly weighed and measured, and their feed consumption was monitored. Additionally, to investigate potential health effects, after 72 weeks of the exposure to ZEN, the fishes' blood was analyzed for major hematological and biochemical indices, and their head kidney, spleen, and liver were examined for morphological, histopathological, cytological, and molecular changes. Finally, to gain insight into the metabolism and distribution of ZEN in fish, the content of free and glucuronidated forms of ZEN and its major metabolites was measured in the intestine, liver, and muscles of the exposed fish. The feed-borne exposure of rainbow trout to ZEN at a dose of 2 mg⋅kg-1 feed resulted in higher feeding efficiency and growth rate, most probably due to the anabolic properties of the ZEN metabolite. Importantly for the consumers of fish, despite absorption and metabolism of ZEN in the digestive system of the fish that had been exposed for 72 weeks, the residuals of ZEN were not transferred to the fishes' muscles, which rules out a potential risk to human health related to the consumption of fish meat. However, the increased growth of fish fed with the contaminated feed may come at some cost, as the exposure to ZEN was associated with modulation of key components of the adaptive and innate immune systems. Moreover, the trunk kidney of ZEN-fed fish showed massive inflammation that was likely caused by pathogen infection. These findings raise concerns about fish health under the current recommended EC guidance values.

Intraperitoneal exposure of whitefish to microcystin-LR induces rapid liver injury followed by regeneration and resilience to subsequent exposures.

To date, there has been no systematic approach comprehensively describing the sequence of pathological changes in fish during prolonged exposure to microcystin-LR (MC-LR). Towards this aim, juvenile whitefish individuals received an intraperitoneal injection with pure MC-LR, and the injection was repeated every week to maintain continuous exposure for 28days. During the exposure period, growth and condition of the fish were assessed based on biometric measurements. Additionally, selected biochemical markers were analysed in the fishes' blood, and their livers were carefully examined for morphological, ultrastructural, and molecular changes. The higher dose of MC-LR (100µg·kg-1) caused severe liver injury at the beginning of the exposure period, whereas the lower dose (10µg·kg-1) caused less, probably reversible injury, and its effects began to be observed later in the exposure period. These marked changes were accompanied by substantial MC-LR uptake by the liver. However, starting on the 7th day of exposure, cell debris began to be removed by phagocytes, then by 14th day, proliferation of liver cells had markedly increased, which led to reconstruction of the liver parenchyma at the end of the treatment. Surprisingly, despite weekly-repeated intraperitoneal injections, MC-LR did not accumulate over time of exposure which suggests its limited uptake in the later phase of exposure. In support, mRNA expression of the membrane transport protein oatp1d was decreased at the same time as the regenerative processes were observed. Our study shows that closing of active membrane transport may serve as one defence mechanism against further MC-LR intoxication.

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

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.

miR-34a and bcl-2 expression in whitefish (Coregonus lavaretus) after microcystin-LR exposure.

Studies on mammals have demonstrated that the expression of miR-34a is associated with process of apoptosis in many cell types, by lowering expression of the anti-apoptotic protein Bcl-2. Despite the role of miR-34a, there is no data about the miR-34a:Bcl-2 interaction in lower vertebrates, especially in fish. In the current study, we determined the nucleotide sequence of miR-34a precursor, predicted its secondary structure, and shed light on the potential role of p53 in activation of miR-34a in whitefish, a salmonid fish species. In parallel, we determined a cDNA sequence of whitefish bcl-2, and gained insight into the primary structure and evolutionary relationship of the whitefish Bcl-2 protein that it codes for. In particular, we were interested whether whitefish bcl-2 3'UTR contains an active target site for miR-34a. Using a computational approach followed by luciferase reporter assay, we confirmed the direct interaction of miR-34a with the whitefish bcl-2 3'UTR. Therefore, we further investigated whether bcl-2 silencing via miR-34a occurs in liver samples of whitefish exposed for 48h to microcystin-LR (MC-LR), a known hepatotoxin and tumor promoter. We noticed a statistically unsignificant up-regulation of miR-34a expression, which was accompanied by a marginally significant increase of bcl-2 mRNA level and the significant increase of bax (pro-apoptotic) mRNA level. However, we found no significant correlation between bcl-2 and miR-34a expression in vivo, which suggests that their involvement in hepatocyte cell responses to MC-LR in whitefish is still questionable.