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

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 n...

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Autores principales: Fjeldbo, Christina S., Aarnes, Eva-Katrine, Malinen, Eirik, Kristensen, Gunnar B., Lyng, Heidi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887009/
https://www.ncbi.nlm.nih.gov/pubmed/27244197
http://dx.doi.org/10.1371/journal.pone.0156259
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author Fjeldbo, Christina S.
Aarnes, Eva-Katrine
Malinen, Eirik
Kristensen, Gunnar B.
Lyng, Heidi
author_facet Fjeldbo, Christina S.
Aarnes, Eva-Katrine
Malinen, Eirik
Kristensen, Gunnar B.
Lyng, Heidi
author_sort Fjeldbo, Christina S.
collection PubMed
description 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 A(Brix) 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 (A(Brix)) 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 A(Brix) (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.
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spelling pubmed-48870092016-06-10 Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients Fjeldbo, Christina S. Aarnes, Eva-Katrine Malinen, Eirik Kristensen, Gunnar B. Lyng, Heidi PLoS One Research Article 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 A(Brix) 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 (A(Brix)) 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 A(Brix) (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. Public Library of Science 2016-05-31 /pmc/articles/PMC4887009/ /pubmed/27244197 http://dx.doi.org/10.1371/journal.pone.0156259 Text en © 2016 Fjeldbo et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Fjeldbo, Christina S.
Aarnes, Eva-Katrine
Malinen, Eirik
Kristensen, Gunnar B.
Lyng, Heidi
Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients
title Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients
title_full Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients
title_fullStr Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients
title_full_unstemmed Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients
title_short Identification and Validation of Reference Genes for RT-qPCR Studies of Hypoxia in Squamous Cervical Cancer Patients
title_sort identification and validation of reference genes for rt-qpcr studies of hypoxia in squamous cervical cancer patients
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887009/
https://www.ncbi.nlm.nih.gov/pubmed/27244197
http://dx.doi.org/10.1371/journal.pone.0156259
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