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

Abstract

The p16INK4a 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 p16INK4a protein can also repress the activity of other transcription factors, such as c-myc, nuclear factorkappaB and c-Jun/AP1. Here, we report that, in two p16WT and p53 WT cell lines (MCF7 and U87), p16INK4a overexpression induces a dramatic decrease in CDK1 protein expression. In response to p16 INK4a, 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 p16INK4a could regulate CDK1 expression at the post-transcriptional level. This CDK1 downregulation is mediated by the 3 0 -untranslated region (3 0 UTR) 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 p16 INK4a-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 CDK1-3 0 UTR 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 p16 INK4a . We suggest that p16INK4a may regulate gene expression by modifying the functional equilibrium of transcription factors and consequently the expression balance of miRNAs.

MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia

“…This observation is surprising because previously published data suggested that miR-650 is expressed independently of immunoglobulin genes. [12][13][14][15] We subsequently performed a bioinformatical analysis of the upstream region (500 nt) of the miR-650 and IgL leader exon by search for Polymerase II promoter sequence (PROSCAN 18 ) and transcription elements (TESS System 19 ). This confirmed that the IgL and miR-650 gene likely use the same major promoter region Table 1.…”

“…However, expression of miR-650 is clearly not strictly dependent on IgL promoter, since it is expressed in all CLL cells and also in other cell types. [12][13][14][15] This suggests that IgL promoter rather serves as a potent enhancer element for miR-650. It is known that, similarly to miR-650, a fraction of microRNAs is located in exons of protein-coding genes, 20 but the data about the regulation of their expression is limited.…”

“…[12][13][14][15] This prompted us to study the potential role of miR-650 for CLL and B cells. Initially we tested the relation of miR-650 levels to CLL prognosis, which at least partially reflects the biology of CLL B cells.…”

“…10 This miRNA was initially identified by a miRAGE cloning approach in colorectal cells, 11 and it seemed to be transcribed independently of the immunoglobulin host gene. 10,[12][13][14] Remarkably, recent studies describe the role of miR-650 in the biology of colorectal cancer, breast cancer, gastric cancer, and melanoma. [12][13][14][15] Here we have determined that miR-650 expression is associated with CLL prognosis, influences B-cell proliferation, and we identify its targets.…”

“…10,[12][13][14] Remarkably, recent studies describe the role of miR-650 in the biology of colorectal cancer, breast cancer, gastric cancer, and melanoma. [12][13][14][15] Here we have determined that miR-650 expression is associated with CLL prognosis, influences B-cell proliferation, and we identify its targets. Moreover, we show that miR-650 expression is regulated by coupled expression with its host gene for IgL.…”