Reference Genes for Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic Studies.
Reference Genes for Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic Studies.
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DOI：
10.1371/journal.pone.0166120
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Ferreira JA
中科院分区：
文献类型：
--
作者：
Lima L;Gaiteiro C;Peixoto A;Soares J;Neves M;Santos LL;Ferreira JA
高度侵袭性的、快速生长的肿瘤由于血液供应不足和/或不规则而含有显著的缺氧或缺氧区域。在缺氧过程中，肿瘤细胞承受了一系列的适应性反应，包括向无氧代谢的转变和转录组的重新编程。低氧诱导因子1（HIF-1）是低氧应答的主要介导因子之一，其在低氧下的稳定性作为致癌刺激物促进化疗抗性、侵袭和转移。通过qRT-PCR进行的基因表达分析是癌细胞表型表征的有力工具。然而，由于细胞经历严重的转录组对缺氧的反应，参考基因的精确鉴定对缺氧研究构成了重大挑战。remodeling.in在此，我们的目的是建立最好的参考基因，研究缺氧对膀胱癌细胞的影响。因此，使用了三种膀胱癌细胞系（T24、5637和HT 1376），其代表了两种不同的侵袭性癌症的致癌途径（FGFR 3/CCND 1和E2 F3/RB 1）。此外，我们已经探索了最合适的控制基因，当解决去铁胺甲磺酸盐（DFX）的影响，铁螯合剂，通常用于避免蛋白酶体降解的HIF-1α，作为缺氧模拟剂。使用生物信息学工具（GeNorm和Normalization），我们从一组7个假定候选基因（HPRT、ACTB、18 S、GAPDH、TBP、B2 M和SDHA）中选出B2 M和HPRT作为所有细胞系和实验条件下最稳定的基因。这些观察结果为未来研究低氧，特别是HIF-1α在膀胱癌细胞中的作用奠定了分子基础。
Highly aggressive, rapidly growing tumors contain significant areas of hypoxia or anoxia as a consequence of inadequate and/or irregular blood supply. During oxygen deprivation, tumor cells withstand a panoply of adaptive responses, including a shift towards anaerobic metabolism and the reprogramming of the transcriptome. One of the major mediators of the transcriptional hypoxic response is the hypoxia-inducible factor 1 (HIF-1), whose stabilization under hypoxia acts as an oncogenic stimulus contributing to chemotherapy resistance, invasion and metastasis. Gene expression analysis by qRT-PCR is a powerful tool for cancer cells phenotypic characterization. Nevertheless, as cells undergo a severe transcriptome remodeling.in response to oxygen deficit, the precise identification of reference genes poses a significant challenge for hypoxic studies. Herein, we aim to establish the best reference genes for studying the effects of hypoxia on bladder cancer cells. Accordingly, three bladder cancer cell lines (T24, 5637, and HT1376) representative of two distinct carcinogenesis pathways to invasive cancer (FGFR3/CCND1 and E2F3/RB1) were used. Additionally, we have explored the most suitable control gene when addressing the influence of Deferoxamine Mesilate salt (DFX), an iron chelator often used to avoid the proteasomal degradation of HIF-1α, acting as an hypoxia-mimetic agent. Using bioinformatics tools (GeNorm and NormFinder), we have elected B2M and HPRT as the most stable genes for all cell lines and experimental conditions out of a panel of seven putative candidates (HPRT, ACTB, 18S, GAPDH, TBP, B2M, and SDHA). These observations set the molecular basis for future studies addressing the effect of hypoxia and particularly HIF-1α in bladder cancer cells.
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