Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients

• Published in: PloS one Vol. 11; no. 5; p. e0156259
• Main Authors: Fjeldbo, Christina S, Aarnes, Eva-Katrine, Malinen, Eirik, Kristensen, Gunnar B, Lyng, Heidi
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 31.05.2016; Public Library of Science (PLoS)
• Subjects: Adult; Aged; Aged, 80 and over; Anoxia; Apoptosis Regulatory Proteins - genetics; Biology; Biology and Life Sciences; Biomarkers; Biomarkers, Tumor - genetics; Cancer; Cancer therapies; Carcinoma, Squamous Cell - complications; Carcinoma, Squamous Cell - genetics; Care and treatment; Cell Line, Tumor; Cervical cancer; Cohort Studies; Complications and side effects; Data processing; Development and progression; Female; Gene Expression; Genes; Genetic aspects; Genomics; Glycoproteins - genetics; Hospitals; Humans; Hypoxia; Hypoxia - complications; Hypoxia - genetics; Identification and classification; Intercellular Signaling Peptides and Proteins - genetics; Lung cancer; Lymph nodes; Lymphatic system; Magnetic resonance; Magnetic resonance imaging; Medical prognosis; Medicine and Health Sciences; Membrane Proteins - genetics; Metastasis; Middle Aged; Nuclear Proteins - genetics; Oncogenes; Parameters; Patients; Physiological aspects; Prognosis; Prostate cancer; Radiation therapy; Research and Analysis Methods; Reverse Transcriptase Polymerase Chain Reaction; Reverse transcription; Serine-Arginine Splicing Factors - genetics; Studies; Subgroups; Superoxide dismutase; Tumor cell lines; Tumors; Uterine Cervical Neoplasms - complications; Uterine Cervical Neoplasms - genetics; Young Adult; United States; Norway
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0156259

Hypoxia is an adverse factor in cervical cancer, and hypoxia-related gene expression could be a powerful biomarker for identifying the aggressive hypoxic tumors. Reverse transcription quantitative PCR (RT-qPCR) is a valuable method for gene expression studies, but suitable reference genes for data normalization that are independent of hypoxia status and clinical parameters of cervical tumors are lacking. In the present work, we aimed to identify reference genes for RT-qPCR studies of hypoxia in squamous cervical cancer. From 422 candidate reference genes selected from the literature, we used Illumina array-based expression profiles to identify 182 genes not affected by hypoxia in cervical cancer, i.e. genes regulated by hypoxia in eight cervical cancer cell lines or correlating with the hypoxia-associated dynamic contrast-enhanced magnetic resonance imaging parameter ABrix in 42 patients, were excluded. Among the 182 genes, nine candidates (CHCHD1, GNB2L1, IPO8, LASP1, RPL27A, RPS12, SOD1, SRSF9, TMBIM6) that were not associated with tumor volume, stage, lymph node involvement or disease progression in array data of 150 patients, were selected for further testing by RT-qPCR. geNorm and NormFinder analyses of RT-qPCR data of 74 patients identified CHCHD1, SRSF9 and TMBIM6 as the optimal set of reference genes, with stable expression both overall and across patient subgroups with different hypoxia status (ABrix) and clinical parameters. The suitability of the three reference genes were validated in studies of the hypoxia-induced genes DDIT3, ERO1A, and STC2. After normalization, the RT-qPCR data of these genes showed a significant correlation with Illumina expression (P<0.001, n = 74) and ABrix (P<0.05, n = 32), and the STC2 data were associated with clinical outcome, in accordance with the Illumina data. Thus, CHCHD1, SRSF9 and TMBIM6 seem to be suitable reference genes for studying hypoxia-related gene expression in squamous cervical cancer samples by RT-qPCR. Moreover, STC2 is a promising prognostic hypoxia biomarker in cervical cancer.