Molecular characterization of the cyclin-dependent protein kinase 6 in whitefish (Coregonus lavaretus) and its potential interplay with miR-34a.

Cyclin-dependent protein kinase 6 (CDK6) plays a pivotal role in the regulation of the cell cycle and cell proliferation in mammals, and disruption of its expression by various microRNAs has been implicated in the pathogenesis of multiple human cancers. In mammals, miR-34a acts as a downstream effector of p53, and thus indirectly targets Cdk6, abrogating its effects. However, no studies have been done so far to examine the mechanistic involvement of miR-34a in the silencing of cdk6 in fish. In the present study, we found that the cDNA sequence of whitefish cdk6 has a 3'UTR region that contains a binding site for miR-34a. Using a luciferase reporter assay, we demonstrated that whitefish cdk6 is a direct target of miR-34a in vitro. In order to confirm this relationship in vivo, we measured the miR-34a and cdk6 mRNA expression patterns in the liver of whitefish after short-term (8, 24, and 48 h) and long-term (14 and 28 days) exposure to microcystin-LR (MC-LR), a known hepatotoxin and tumor promoter. In contrast to the in vitro findings, we noticed an up-regulation of miR-34a and cdk6 expression after long-term MC-LR treatment. While these results indicate that both, miR-34a and cdk6 are responsive to MC-LR treatment, they do not support the presence of a miR-34a:cdk6 mRNA regulatory pair in the MC-LR-challanged whitefish liver in vivo. On the other hand, our findings suggests that cell regulatory elements, partnering with either miR-34a or cdk6, are worthy of further screening to better understand the molecular mechanisms that underlie the physiological response of fish challenged with hepatotoxic environmental pollutants like microcystins.