Miikkulainen Petra, Högel Heidi, Rantanen Krista, Suomi Tomi, Kouvonen Petri, Elo Laura L, Jaakkola Panu M
Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Tykistökatu 6, 20520 Turku, Finland.
Department of Medical Biochemistry, Faculty of Medicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland.
Cancer Metab. 2017 Jul 4;5:5. doi: 10.1186/s40170-017-0167-y. eCollection 2017.
A 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.
To 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.
Our 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.
Our findings suggest crucial involvement of PHD3 in the maintenance of key cellular functions including glycolysis and protein synthesis in ccRCC.
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，这导致即使在氧合良好的条件下缺氧诱导因子（HIF）通路也被激活。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在癌症中的几种功能和下游靶点，但PHD3表达升高在ccRCC中的作用尚不清楚。
为了深入了解ccRCC中高PHD3表达的功能，我们在常氧和低氧条件下，使用小干扰RNA（siRNA）敲低786-O细胞中的PHD3，并对纯化的肽样品进行发现质谱分析（LC-MS/MS）。使用肽水平表达变化平均程序对蛋白质组数据进行无标记定量，并随后进行基因本体富集分析，以分析LC-MS/MS结果。
我们的数据揭示了PHD3敲低具有有趣的广泛影响，有91种蛋白质受到显著调节。在低氧条件下，无论是受调节蛋白质的数量还是蛋白质表达水平的范围，对PHD3沉默的反应都比常氧条件下更广泛。受PHD3表达调节的主要细胞功能是葡萄糖代谢、蛋白质翻译和信使核糖核酸（mRNA）加工。PHD3沉默导致从葡萄糖转运到乳酸产生的大多数糖酵解酶下调，细胞外酸化和乳酸产生减少以及细胞耗氧率增加支持了这一点。此外，作为对PHD3沉默的反应，观察到mRNA加工相关蛋白质上调以及一些核糖体蛋白质发生改变。对翻译机制上游效应器的进一步研究揭示了PHD3在调节mTOR通路信号传导中的可能作用。
我们的研究结果表明，PHD3在维持ccRCC的关键细胞功能（包括糖酵解和蛋白质合成）中起关键作用。
