Posttranscriptional gene silencing of p27 in primary prostate epithelial cells by small inhibitory RNA (siRNA)
The cell cycle is an ordered set of events, culminating in cell growth and division into two daughter cells. The cell cycle is regulated by the intricate interplay of many molecules. Cdks (cyclin dependent kinases), along with cyclins, are major control switches for the cell cycle, causing the cell to move from G1 to S or G2 to M. Their function is tightly regulated by Cdk-inhibitors such as the p21 and p27 Cip/Kip proteins. Defects in many of the molecules that regulate the cell cycle have been implicated in cancer development. An important example is the common downregulation of the Cdk inhibitor p27 in prostate cancers. p27/Kip1 regulates the cell cycle by inhibiting the checkpoint kinase Cdk2/cyclin E kinase and blocking cell cycle progression in G1. In prostate cancer, reduced levels of p27 protein are associated with clinically aggressive tumors and poor prognosis. However, the molecular mechanisms of how the decrease in p27 levels contributes to prostate tumorigenesis are still poorly understood. The aim of this study was to establish a system to downregulate p27 protein in primary diploid prostate epithelial cells (PrEC) for prolonged periods in order to create a model for further studies on its potential role in prostate cancer. In the experiments described here, sustained long term knockdown of p27 was achieved in human primary prostate cells by designing a lentivirus based vector system to deliver p27 specific siRNA. Immunoblotting showed a striking though not complete downregulation of p27 in PrEC. This may imitate p27 downregulation in tumorigenesis even better than a complete genetic knockout as the p27 gene is virtually never homozygously inactivated in prostate cancers. Thus, this system should provide a suitable model for future studies addressing the potential role of p27 in human prostate cancer development.
siRNA, PrEC, p27, Zellzyklus, Prostate Cancer, Posttranscriptional Gene Silencing
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