title: miR-34a and bcl-2 expression in whitefish (Coregonus lavaretus) after microcystin-LR exposure.
abstract: 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.
authors.affiliation: Department of Environmental Biotechnology, Faculty of Environmental Sciences, University of Warmia and Mazury in Olsztyn, ul. Słoneczna 45G, 10-709 Olsztyn, Poland. Electronic address: alicja.lakomiak@uwm.edu.pl.
authors.forename: Alicja
authors.initials: A
authors.lastname: Łakomiak
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: Paweł
authors.initials: P
authors.lastname: Brzuzan
authors.affiliation: Division 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: Ewa
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: Maciej
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: Maciej
authors.initials: M
authors.lastname: Woźny
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019295:Computational Biology; D017124:Conserved Sequence; D005786:Gene Expression Regulation / Q000187:drug effects*; D057809:HEK293 Cells; D006801:Humans; D008387:Marine Toxins; D035683:MicroRNAs / Q000235:genetics*; D052998:Microcystins / Q000494:pharmacology*; D008969:Molecular Sequence Data; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012487:Salmonidae / Q000235:genetics*; D013997:Time Factors
chemical_list: D020413:3' Untranslated Regions; D008387:Marine Toxins; D035683:MicroRNAs; D052998:Microcystins; D019253:Proto-Oncogene Proteins c-bcl-2; C057862:cyanoginosin LR
keywords: Fish; Reporter analysis; bax; bcl-2; miR-34a