BackgroundA key feature of clear cell renal cell carcinoma (ccRCC) is the inactivation of the von Hippel-Lindau tumour suppressor protein (pVHL) that leads to the activation of hypoxia-inducible factor (HIF) pathway also in well-oxygenated conditions. Important regulator of HIF-α, prolyl hydroxylase PHD3, is expressed in high amounts in ccRCC. Although several functions and downstream targets for PHD3 in cancer have been suggested, the role of elevated PHD3 expression in ccRCC is not clear.MethodsTo gain insight into the functions of high PHD3 expression in ccRCC, we used PHD3 knockdown by siRNA in 786-O cells under normoxic and hypoxic conditions and performed discovery mass spectrometry (LC-MS/MS) of the purified peptide samples. The LC-MS/MS results were analysed by label-free quantification of proteome data using a peptide-level expression-change averaging procedure and subsequent gene ontology enrichment analysis.ResultsOur data reveals an intriguingly widespread effect of PHD3 knockdown with 91 significantly regulated proteins. Under hypoxia, the response to PHD3 silencing was wider than under normoxia illustrated by both the number of regulated proteins and by the range of protein expression levels. The main cellular functions regulated by PHD3 expression were glucose metabolism, protein translation and messenger RNA (mRNA) processing. PHD3 silencing led to downregulation of most glycolytic enzymes from glucose transport to lactate production supported by the reduction in extracellular acidification and lactate production and increase in cellular oxygen consumption rate. Moreover, upregulation of mRNA processing-related proteins and alteration in a number of ribosomal proteins was seen as a response to PHD3 silencing. Further studies on upstream effectors of the translational machinery revealed a possible role for PHD3 in regulation of mTOR pathway signalling.ConclusionsOur findings suggest crucial involvement of PHD3 in the maintenance of key cellular functions including glycolysis and protein synthesis in ccRCC.

中文翻译：

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HIF 脯氨酰羟化酶 PHD3 调节透明细胞肾细胞癌中的翻译机制和葡萄糖代谢


背景透明细胞肾细胞癌 (ccRCC) 的一个关键特征是 von Hippel-Lindau 肿瘤抑制蛋白 (pVHL) 失活，导致缺氧诱导因子 (HIF) 通路在氧合良好的条件下也被激活。 HIF-α 的重要调节因子脯氨酰羟化酶 PHD3 在 ccRCC 中大量表达。尽管已经提出了 PHD3 在癌症中的多种功能和下游靶标，但 PHD3 表达升高在 ccRCC 中的作用尚不清楚。方法为了深入了解 PHD3 高表达在 ccRCC 中的功能，我们在 786-O 细胞中通过 siRNA 敲低 PHD3在常氧和低氧条件下对纯化的肽样品进行发现质谱 (LC-MS/MS)。使用肽水平表达变化平均程序和随后的基因本体富集分析，通过对蛋白质组数据进行无标记定量来分析 LC-MS/MS 结果。结果我们的数据揭示了 PHD3 敲低对 91 种显着调节的蛋白质的广泛影响。在缺氧条件下，对 PHD3 沉默的反应比常氧条件下更广泛，这可以通过调节蛋白的数量和蛋白表达水平的范围来说明。 PHD3表达调节的主要细胞功能是葡萄糖代谢、蛋白质翻译和信使RNA（mRNA）加工。 PHD3沉默导致大多数糖酵解酶从葡萄糖转运到乳酸生产的下调，这受到细胞外酸化和乳酸生产减少以及细胞耗氧率增加的支持。此外，mRNA 加工相关蛋白的上调和许多核糖体蛋白的改变被视为对 PHD3 沉默的反应。 对翻译机制上游效应器的进一步研究揭示了 PHD3 在 mTOR 通路信号传导调节中的可能作用。结论我们的研究结果表明 PHD3 在维持关键细胞功能（包括 ccRCC 中的糖酵解和蛋白质合成）中发挥着重要作用。