Introduction: Hypoxic cancers are radioresistant, but biomarkers based on expression of multiple genes can identify patients who will benefit from hypoxia modification. Most studies identifying relevant genes exposed cells in culture to 1% oxygen, which activates hypoxia-inducible factor (HIF). However, oxygen concentrations in hypoxic tumours are heterogeneous ranging from <0.1%. As lower oxygen levels would likely affect transcriptional responses, we aimed to investigate how gene selection at different oxygen levels affects the genes identified and their prognostic capability. Methods: Four MIBC (J82, T24, UMUC3, HT1376) and two non-MIBC (RT4, RT112) bladder cancer cell lines were exposed to varying oxygen levels (20%, 1%, 0.2% and 0.1% O2) for 24 h and were then harvested and frozen. RNA was extracted and transcriptomes analysed using Clariom S microarrays. Differences in gene expression were investigated. Prognostic and predictive significance of a published 24-gene signature was compared with one generated from genes identified at lower oxygen levels. Results: The number of upregulated genes increased with decreasing O2level. The number of biological pathways involved also increased. Differences between cell lines dominated those due to hypoxia. Some genes were commonly upregulated in MIBC and NMIBC cells and others increased exclusively in either MIBC or NMIBC cells. The median expression of a published 24-gene bladder cancer hypoxia-associated signature increased with decreasing oxygen levels. Seventy-seven genes were upregulated in at least three cell lines by exposure to 0.1% O2. The median expression of the 77 genes was of borderline prognostic significance in the bladder cancer cohort in the TCGA (The Cancer Genome Atlas). Five of the seventy-seven genes upregulated by hypoxia were present in the twenty-four-gene bladder hypoxia signature. The median expression of the 5 genes demonstrated identical prognostication to the 24-gene signature but failed to predict benefit from hypoxia modification. Conclusions: The number of genes upregulated by exposure of bladder cancer cells to hypoxia increases as O2level is decreased from 1% to 0.2% to 0.1%. Differential upregulation of gene expression by MIBC and NMIBC cells and the associated biological pathways may be useful in understanding the genetics of bladder cancer invasiveness. Based on a search of the literature, this is the first study that assessed the expression of genes in bladder cancer using three hypoxic concentration levels to identify biomarkers for disease progression and prognosis among differentially expressed bladder cancer genes.
引言：缺氧性癌症具有放射抗性，但基于多基因表达的生物标志物可识别出能从缺氧修饰治疗中获益的患者。多数相关基因研究采用1%氧浓度处理培养细胞以激活缺氧诱导因子（HIF）。然而缺氧肿瘤中的氧浓度存在异质性，可低至<0.1%。由于更低氧浓度可能影响转录反应，本研究旨在探讨不同氧浓度水平下的基因选择如何影响所识别基因及其预后预测能力。 方法：将四种肌层浸润性膀胱癌（MIBC）细胞系（J82、T24、UMUC3、HT1376）和两种非肌层浸润性膀胱癌（NMIBC）细胞系（RT4、RT112）分别暴露于不同氧浓度（20%、1%、0.2%和0.1% O2）24小时后收集冻存。提取RNA并使用Clariom S微阵列分析转录组，探究基因表达差异。将已发表的24基因标志物的预后与预测价值，与在更低氧浓度下识别基因构建的标志物进行比较。 结果：上调基因数量随氧浓度降低而增加，涉及的生物学通路也随之增多。细胞系间差异对基因表达的影响超过缺氧效应。部分基因在MIBC和NMIBC细胞中共同上调，另一些则仅在MIBC或NMIBC细胞中特异性上调。已发表的24基因膀胱癌缺氧相关标志物的中位表达量随氧浓度降低而增加。暴露于0.1% O2时，77个基因在至少三种细胞系中上调。在癌症基因组图谱（TCGA）膀胱癌队列中，这77个基因的中位表达量具有临界预后意义。其中5个缺氧上调基因存在于24基因膀胱缺氧标志物中，这5个基因的中位表达量与24基因标志物具有同等预后价值，但未能预测缺氧修饰治疗的获益。 结论：膀胱癌细胞在缺氧环境下（氧浓度从1%降至0.2%再到0.1%）的上调基因数量逐步增加。MIBC与NMIBC细胞基因表达的差异性上调及其相关生物学通路，可能有助于理解膀胱癌侵袭性的遗传学机制。据文献检索，本研究首次采用三种缺氧浓度评估膀胱癌基因表达，旨在从差异表达基因中筛选疾病进展和预后的生物标志物。
Gene Expression in Muscle-Invasive and Non-Muscle-Invasive Bladder Cancer Cells Exposed to Hypoxia
肿瘤(癌症)患者之家