title: c-myc in whitefish (Coregonus lavaretus): structure, expression, and insights into possible posttranscriptional regulatory mechanism.
abstract: 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.
authors.affiliation: Department of Environmental Biotechnology, Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, ul. Słoneczna 45G, 10-709, Olsztyn, Poland. brzuzan@uwm.edu.pl.
authors.forename: P
authors.initials: P
authors.lastname: Brzuzan
authors.affiliation: Department of Environmental Biotechnology, Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, ul. Słoneczna 45G, 10-709, Olsztyn, Poland.
authors.forename: C
authors.initials: C
authors.lastname: Kramer
authors.affiliation: Department of Environmental Biotechnology, Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, ul. Słoneczna 45G, 10-709, Olsztyn, Poland.
authors.forename: A
authors.initials: A
authors.lastname: Łakomiak
authors.affiliation: Department of Veterinary Prevention and Feed Hygiene, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, ul. Oczapowskiego 13, 10-950, Olsztyn, Poland.
authors.forename: E
authors.initials: E
authors.lastname: Jakimiuk
authors.affiliation: Department of Environmental Biotechnology, Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, ul. Słoneczna 45G, 10-709, Olsztyn, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Florczyk
authors.affiliation: Department of Environmental Biotechnology, Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, ul. Słoneczna 45G, 10-709, Olsztyn, Poland.
authors.forename: M
authors.initials: M
authors.lastname: Woźny
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005786:Gene Expression Regulation / Q000502:physiology*; D016259:Genes, myc / Q000235:genetics / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D008387:Marine Toxins; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D052998:Microcystins / Q000633:toxicity; D008969:Molecular Sequence Data; D010802:Phylogeny; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D017858:Salmoniformes / Q000378:metabolism*
chemical_list: D008387:Marine Toxins; D035683:MicroRNAs; D052998:Microcystins; C057862:cyanoginosin LR
keywords: MicroRNA; Microcystin-LR; Reporter analysis; c-myc; miR-34a