Koritzinsky Marianne, Seigneuric Renaud, Magagnin Michaël G, van den Beucken Twan, Lambin Philippe, Wouters Bradly G
Department of Radiation Oncology (MAASTRO Lab), Research Institute Growth and Development, Maastricht, The Netherlands.
Radiother Oncol. 2005 Aug;76(2):177-86. doi: 10.1016/j.radonc.2005.06.036.
Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined.
DU145 prostate carcinoma cells were exposed to 4h of hypoxia (<0.02% O2). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR.
One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity.
Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia.
缺氧会导致mRNA翻译效率迅速降低。这种抑制作用对所有mRNA种类的影响程度不尽相同,因此可能在很大程度上导致缺氧诱导的蛋白质表达差异。本研究的目的是描述急性缺氧期间基因表达的变化,并评估通过mRNA翻译进行的调控对这些变化的作用。对于每个基因,确定了mRNA丰度变化与mRNA翻译变化的作用。
将DU145前列腺癌细胞暴露于缺氧环境(氧气含量<0.02%)4小时。通过蔗糖梯度沉降分离高效翻译的mRNA。使用Affymetrix微阵列技术评估转录和翻译对基因表达的作用。结果通过定量PCR进行验证。
与转录水平仅76个基因相比,在高效翻译的mRNA组分中,有120个基因因缺氧而上调超过4倍。在翻译变化最大的50个基因中,有11个基因在翻译组分中的表达相对于其整体转录水平高出2倍以上。对其诱导具有最高翻译作用的基因是CITED-2,它是HIF-1转录活性的负调节因子。
mRNA翻译的基因特异性调控在很大程度上导致了缺氧期间的基因表达差异。