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

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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.

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Acknowledgments

We thank Dr. Stefan Dobosz from the Department of the Salmonid Research in Rutki (Inland Fisheries Institute in Olsztyn, Poland) for his excellent technical assistance during hatchery operations. We also thank Elżbieta Wojdat, M.Sc., from the Institute of Immunology and Experimental Therapy in Wrocław, Polish Academy of Sciences, for her valuable technical assistance in HEK293 cell culture procedures. The authors wish to thank Trev Hill and Mark Leonard, M.Sc., for proofreading the manuscript. The study was supported by research grant from National Science Centre (Poland) No. DEC-2012/07/B/NZ9/01320.

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Authors and Affiliations

  1. Department of Environmental Biotechnology, Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, ul. Słoneczna 45G, 10-709, Olsztyn, Poland

    P. Brzuzan, C. Kramer, A. Łakomiak, M. Florczyk & M. Woźny

  2. 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

    E. Jakimiuk

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  1. P. Brzuzan
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Correspondence to P. Brzuzan.

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Brzuzan, P., Kramer, C., Łakomiak, A. et al. c-myc in whitefish (Coregonus lavaretus): structure, expression, and insights into possible posttranscriptional regulatory mechanism. Fish Physiol Biochem 41, 1155–1171 (2015). https://doi.org/10.1007/s10695-015-0077-2

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