Cyclin-dependent kinase 1 expression is inhibited by p16INK4a at the post-transcriptional level through the microRNA pathway

The p(16INK4a) protein regulates cell cycle progression mainly by inhibiting the activity of G1-phase cyclin-dependent kinases (CDKs) 4 and 6, the subsequent retinoblastoma protein (pRb) phosphorylation and E2F transcription factor release. The p(16INK4a) protein can also repress the activity of other transcription factors, such as c-myc, nuclear factor-kappaB and c-Jun/AP1. Here, we report that, in two p16(-/-), pRb(WT) and p53(WT) cell lines (MCF7 and U87), p(16INK4a) overexpression induces a dramatic decrease in CDK1 protein expression. In response to p(16INK4a), the decreased rate of CDK1 protein synthesis, its unchanged protein half- life, unreduced CDK1 mRNA steady- state levels and mRNA half- life allow us to hypothesize that p(16INK4a) could regulate CDK1 expression at the post- transcriptional level. This CDK1 downregulation is mediated by the 30-untranslated region (30UTR) of CDK1 mRNA as shown by translational inhibition in luciferase assays and is associated with a modified expression balance of microRNAs (miRNAs) that potentially regulate CDK1, analyzed by TaqMan Human microRNA Array. The p16INK4a-induced expression of two miRNAs (miR-410 and miR-650 chosen as an example) in MCF7 cells is confirmed by individual reverse transcriptionqPCR. Furthermore, we show the interaction of miR-410 or miR-650 with CDK1-30UTR by luciferase assays. Endogenous CDK1 expression decreases upon both miRNA overexpression and increases with their simultaneous inhibition. The induction of miR-410, but not miR-650 could be related to the pRb/ E2F pathway. These results demonstrate the post-transcriptional inhibition of CDK1 by p(16INK4a). We suggest that p(16INK4a) may regulate gene expression by modifying the functional equilibrium of transcription factors and consequently the expression balance of miRNAs. Oncogene (2011) 30, 1880-1891; doi: 10.1038/onc. 2010.570; published online 20 December 2010