Peking University, Beijing, Apr. 21, 2010: Researchers from Peking University have made several breakthroughs in the field of life sciences, including a new mechanism in p53 (one tumor suppressor) activation mediated by ARF and the important role miRNAs plays in the flowing regulation of cyclin D1expression and endothelial proliferation. These research results were recently published in top journals of that field, Nature and PNAS .
In the first treatise, researchers reveal a new mechanism in p53 activation mediated by ARF and a new kind of enzyme and illustrate the role this new enzyme plays in p53 activation.
Tumor suppressors are the kind of gene preventing cells from excessive growth and proliferation to suppress tumorigenesis. For normal human cells, coordination between growth-promoting genes and growth-controlling ones is a significant molecular mechanism in determining the growth of cells. Until now, there have been 10 defined tumor suppressors among which p53 is a better known one. It is titled ‘Defender of Genes’ because it plays an important part in sustaining normal growth of cells and suppressing vicious proliferation of them.
Under oncogenic stress, p53 activation needs a tumor suppressor gene called ARF. Recent studies showed that p53 activation mediated by ARF, but not that induced by DNA damage, acts as a major protection against tumorigenesis, suggesting that ARF-p53 axis has more fundamental functions in tumor suppression than originally thought.
Because ARF is a very stable protein in most human cell lines, scientists hold that ARF induction is mediated mainly at the transcriptional level and that activation of the ARF-p53 pathway is a much slower and largely irreversible process by comparison with p53 activation after DNA damage.
In this treatise, researchers find that ARF is very unstable in normal human cells but that its degradation is inhibited in cancerous cells. Through biochemical purification, they identified a specific ubiquitin ligase for ARF and named it ULF. ULF interacts with ARF both in vitro and in vivo and promotes degradation of ARF. The experiment of ULF knockdown confirms that ULF knockdown stabilizes ARF in normal human cells.
Moreover, it is discovered that NPM and c-Myc, both of which are commonly overexpressed proteins in cancer cells, are able to abrogate ULF-mediated ARF ubiquitylation, and therefore promote ARF stabilization in cancer cells. These findings reveal the dynamic feature of the ARF-p53 pathway and illustrate that transcription-independent mechanisms are of critical importance to ARF regulation.
In the second treatise, researchers demonstrate for the first time that laminar shear in veins regulates the expression of miRNA and miR-19a, one of the miRNAs, plays an important role in the flowing regulation of cyclin D1 expression and endothelial proliferation.
If there are alterations in biological, chemical, and physical properties in the flowing blood, vascular endothelium is usually capable of sensing them. Shear stress, the frictional force created by blood flow, exerts a variety of effects on endothelial structure and function. Endothelial cells respond to the changes in mechanical forces and later results in modulation of molecular signaling and cell functions. Suppression of endothelial cells by steady laminar flow is one of the examples. But the molecular mechanisms by which endothelial cells respond to changes in mechanical properties of blood flow and undergo structural and functional adaptations remain to be elucidated. Emerging evidence supports the concept that shear stress exerts its physiological effects by modulating the gene expression of endothelial cells.
Using the miRNA microarray approach, researchers compared the miRNA expression profiles in human umbilical vein endothelial cells (HUVECs) following 12 h of laminar shear stress at 12 dyn/cm2 with those cultured under static condition. Among 569 individual miRNAs, 35 miRNAs were found to be significantly up-regulated and 26 were significantly down-regulated in the laminar flow-treated HUVECs in comparison with the static control cells. Among the miRNAs, miR-19a was expressed at a high abundance under static condition and its level was greatly increased after 12 h of shear stress exposure.
After that, in order to select the target spot regulated by miRNAs, the researchers made up the cell line of statically over-expressed miR-19a. A number of analyses demonstrate that stable transfection of miR-19a significantly decreased the expression of a reporter gene (this reporter gene is controlled by a conserved 3′-untranslated region of the cyclinD1 gene) and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. This suppressive effect was diminished when the endogenous miR-19a was inhibited.
The main discovery in this study is that cyclin D1 is a target of miR-19a in endothelial cells, which was confirmed by the analysis of reporter genes and Western blotting. More importantly, they’ve discovered that shear inducement is essential to suppress the level of cyclin D1 expression. Old discoveries show that laminar shear increases the transcription of cyclin D1 and arrests cell cycle, which this new research provides with a possible explanation.
Translated by: Zhang Chunlan
Edited by: Seren
Source: PKU News (Chinese)
