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.
Keywords: Fish; Reporter analysis; bax; bcl-2; miR-34a.
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