Abstract
MicroRNAs (miRs) are a large class of small regulatory RNAs that function as nodes of signaling networks. This implicates that miRs expression has to be finely tuned, as observed during cell cycle progression. Here, using an expression profiling approach, we provide evidence that the CDK inhibitor p27Kip1 regulates miRs expression following cell cycle exit. By using wild type and p27KO cells harvested in different phases of the cell cycle we identified several miRs regulated by p27Kip1 during the G1 to S phase transition. Among these miRs, we identified miR-223 as a miR specifically upregulated by p27Kip1 in G1 arrested cells. Our data demonstrate that p27Kip1 regulated the expression of miR-223, via two distinct mechanisms. p27Kip1 directly stabilized mature miR-223 expression, acting as a RNA binding protein and it controlled E2F1 expression that, in turn, regulated miR-223 promoter activity. The resulting elevated miR-223 levels ultimately participated to arresting cell cycle progression following contact inhibition. Importantly, this mechanism of growth control was conserved in human cells and deranged in breast cancers. Here, we identify a novel and conserved function of p27Kip1 that, by modulating miR-223 expression, contributes to proper regulation of cell cycle exit following contact inhibition. Thus we propose a new role for miR-223 in the regulation of breast cancer progression.
Highlights
MicroRNAs are a large class of small regulatory RNAs, ~22 nucleotides long, important for the regulation of numerous biological processes [1]
To understand if the regulation of miR-223 by p27 played a role during transformation, we looked at miR223 expression in p27 wild type (p27WT) and p27 knock-out (p27KO) 3T3 fibroblasts transformed with the oncogenic form of K-Ras4B (K-RasV12) (Supplementary Figure 4A), since p27 expression is maintained and has a particular relevance in K-RasV12 transformed cells [29]
We evaluated the expression of p27 and miR-223 in a panel of breast cancer derived cell lines, grown in exponential growth (EG) or highly confluent (HC). p27 was considerably upregulated by contact inhibition only in fibroadenoma-derived MCF10A cells (Figure 6A) and this was paralleled by upregulation of miR-223
Summary
MicroRNAs (miRs) are a large class of small regulatory RNAs, ~22 nucleotides long, important for the regulation of numerous biological processes [1]. Nutrient deprivation and contact inhibition are the most common stimuli that arrest normal cell proliferation and that are lost in cancer. The regulation of p27 following anti-mitogenic stimuli is far less clear and whether the control of cell cycle exit by p27 is based on the regulation of miRs expression is not known. We address this particular aspect, studying the fluctuation of miR levels during cell cycle exit, with particular emphasis on the role played by p27
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