Reference Genes for Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic Studies.
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.
Experimental Pathology and Therapeutics Group - Research Center, Portuguese Institute of Oncology of Porto (IPO-Porto), 4200-072, Porto, Portugal.|i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.|Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-135 Porto, Portugal.
Luís
L
Lima
Experimental Pathology and Therapeutics Group - Research Center, Portuguese Institute of Oncology of Porto (IPO-Porto), 4200-072, Porto, Portugal.
Cristiana
C
Gaiteiro
Experimental Pathology and Therapeutics Group - Research Center, Portuguese Institute of Oncology of Porto (IPO-Porto), 4200-072, Porto, Portugal.|i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.|Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4050-313 Porto, Portugal.
Andreia
A
Peixoto
Experimental Pathology and Therapeutics Group - Research Center, Portuguese Institute of Oncology of Porto (IPO-Porto), 4200-072, Porto, Portugal.
Janine
J
Soares
Experimental Pathology and Therapeutics Group - Research Center, Portuguese Institute of Oncology of Porto (IPO-Porto), 4200-072, Porto, Portugal.|Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4050-313 Porto, Portugal.
Manuel
M
Neves
Experimental Pathology and Therapeutics Group - Research Center, Portuguese Institute of Oncology of Porto (IPO-Porto), 4200-072, Porto, Portugal.|Health School of University of Fernando Pessoa, 4249-004, Porto, Portugal.|Department of Surgical Oncology, Portuguese Institute of Oncology of Porto (IPO-Porto), 4200-072, Porto, Portugal.
Lúcio Lara
LL
Santos
Experimental Pathology and Therapeutics Group - Research Center, Portuguese Institute of Oncology of Porto (IPO-Porto), 4200-072, Porto, Portugal.|i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Portugal.|Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-135 Porto, Portugal.|Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4050-313 Porto, Portugal.
José Alexandre
JA
Ferreira
D000199:Actins / Q000235:genetics; D045744:Cell Line, Tumor; D003676:Deferoxamine / Q000494:pharmacology; D042963:Electron Transport Complex II / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D000860:Hypoxia; D007502:Iron Chelating Agents / Q000494:pharmacology; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction; D035181:TATA-Box Binding Protein / Q000235:genetics; D059467:Transcriptome / Q000187:drug effects / Q000235:genetics*; D001749:Urinary Bladder Neoplasms / Q000235:genetics* / Q000473:pathology; D001613:beta 2-Microglobulin / Q000235:genetics
D000199:Actins; D007502:Iron Chelating Agents; D012337:RNA, Ribosomal, 18S; D035181:TATA-Box Binding Protein; D001613:beta 2-Microglobulin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D042963:Electron Transport Complex II; C511268:SDHA protein, human; D007041:Hypoxanthine Phosphoribosyltransferase; D003676:Deferoxamine