miR-122-5p as a plasma biomarker of liver injury in fish exposed to microcystin-LR.

Recent studies have shown the presence of large amounts of microRNAs (miRNAs; miRs) from damaged cells in the peripheral blood. In this study, we investigated the levels of miRNAs circulating in the blood plasma of whitefish (Coregonus lavaretus) after exposure to microcystin-LR. We used real-time PCR to examine the relative expression of plasma levels of 4 miRNAs (miR-122-5p and let-7c-5p, the liver-enriched microRNAs, miR-148a-3p which promotes the hapatospecific phenotype in mammals, and miR-92a-3p, a cell proliferation and angiogenesis promoter, potentially hepatocarcinogenic) during the first 48 h after exposure to MC-LR. We observed a rapid increase of miR-122-5p levels 8 h after exposure (P < 0.05), which continued to the end of the experiment. Our results demonstrated that the plasma miR-122-5p was indicative of MC-LR-induced liver injury, exhibiting areas under the curve close to 1 in ROC analysis (AUC = 0.976, P < 0.001). Although plasma levels of miR-148a-3p and miR-92a-3p were significantly elevated by the end of the experiment, their discriminative power was lower than reported for the miR-122-5p. Based on these results and reports on miRNA-based diagnosis of liver injuries in mammals, plasma miR-122-5p could be considered as a robust, new generation diagnostic biomarker in fish, helpful for the non-invasive diagnosis of liver damage.

c-myc in whitefish (Coregonus lavaretus): structure, expression, and insights into possible posttranscriptional regulatory mechanism.

c-myc has a crucial function in growth control, differentiation, and apoptosis of vertebrate cells. Despite the important role of c-myc in mediating the biological effects, studies of c-myc gene expression and factors that control it in organisms other than mammals, such as fish, have been rare. In the current study, we asked whether c-myc mRNA of whitefish, a feasible organism for pollution monitoring in aquatic systems and a model in toxicological research, contains activity sites for regulatory motifs in its 5'- and 3'-UTRs, similar to those found in mammals. We were particularly interested in whether miRNA-34, a known negative regulator of c-myc's in mammals, is able to regulate c-myc in fish. To answer these questions, we determined the mRNA sequence of whitefish c-myc and inferred the structure of the protein that it codes for. We found that the active sites of mRNA and structures of the inferred c-myc protein are similar to those found in mammals and other fish. Remarkably, levels of c-myc mRNA expression were very high in ovaries compared to other tissues of whitefish, thus corroborating previous data in fish. Using bioinformatic searches on c-myc 3'-UTR, we confirmed the presence of two miRNA-34a (miR-34a) response elements. Luciferase reporter assay showed that activity of reporters containing either the miR response elements or entire c-myc 3'-UTR was significantly reduced (p < 0.001) by ectopic expression of miR-34a. Therefore, we further investigated possible involvement of miR-34a in c-myc gene silencing by profiling the expression of both genes in livers of whitefish treated for 8, 24, 48 h with MC-LR, a potent c-myc inducer in mammals. Although the difference was only significant at p = 0.08, the expression of c-myc mRNA in challenged whitefish after 24 h of the treatment was notably higher than that in livers of control fish. Concurrently, we noticed slight but significant up-regulation of miR-34a after 24 and 48 h of the challenge (p < 0.05); however, we found no significant correlation of the c-myc mRNA levels and miR-34a expression. Together, these results suggest that miR-34a might regulate c-myc gene expression in whitefish liver; however, their involvement in MC-LR hepatotoxicity should be clarified in future studies.