A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma

Abstract Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible CYGB expression is observed in several cancer cell lines and particularly in...

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Autores principales:	Joey De Backer, Darko Maric, Matthias Bosman, Sylvia Dewilde, David Hoogewijs
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7afe878872f44e2b8088cf0997f2de3e
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spelling	oai:doaj.org-article:7afe878872f44e2b8088cf0997f2de3e2021-12-02T15:49:53ZA reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma10.1038/s41598-021-90284-62045-2322https://doaj.org/article/7afe878872f44e2b8088cf0997f2de3e2021-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-90284-6https://doaj.org/toc/2045-2322Abstract Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible CYGB expression is observed in several cancer cell lines and particularly in various melanoma-derived cell lines. However, reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. Limited evidence exists to support selection of the commonly used reference genes in hypoxic models of melanoma. This study aimed to select the optimal reference genes to study CYGB expression levels in melanoma cell lines exposed to hypoxic conditions (0.2% O2) and to the HIF prolyl hydroxylase inhibitor roxadustat (FG-4592). The expression levels of candidate genes were assessed by qPCR and the stability of genes was evaluated using the geNorm and NormFinder algorithms. Our results display that B2M and YWHAZ represent the most optimal reference genes to reliably quantify hypoxia-inducible CYGB expression in melanoma cell lines. We further validate hypoxia-inducible CYGB expression on protein level and by using CYGB promoter-driven luciferase reporter assays in melanoma cell lines.Joey De BackerDarko MaricMatthias BosmanSylvia DewildeDavid HoogewijsNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Joey De Backer Darko Maric Matthias Bosman Sylvia Dewilde David Hoogewijs A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma
description	Abstract Cytoglobin (CYGB) is a ubiquitously expressed protein with a protective role against oxidative stress, fibrosis and tumor growth, shown to be transcriptionally regulated under hypoxic conditions. Hypoxia-inducible CYGB expression is observed in several cancer cell lines and particularly in various melanoma-derived cell lines. However, reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. Limited evidence exists to support selection of the commonly used reference genes in hypoxic models of melanoma. This study aimed to select the optimal reference genes to study CYGB expression levels in melanoma cell lines exposed to hypoxic conditions (0.2% O2) and to the HIF prolyl hydroxylase inhibitor roxadustat (FG-4592). The expression levels of candidate genes were assessed by qPCR and the stability of genes was evaluated using the geNorm and NormFinder algorithms. Our results display that B2M and YWHAZ represent the most optimal reference genes to reliably quantify hypoxia-inducible CYGB expression in melanoma cell lines. We further validate hypoxia-inducible CYGB expression on protein level and by using CYGB promoter-driven luciferase reporter assays in melanoma cell lines.
format	article
author	Joey De Backer Darko Maric Matthias Bosman Sylvia Dewilde David Hoogewijs
author_facet	Joey De Backer Darko Maric Matthias Bosman Sylvia Dewilde David Hoogewijs
author_sort	Joey De Backer
title	A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma
title_short	A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma
title_full	A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma
title_fullStr	A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma
title_full_unstemmed	A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma
title_sort	reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/7afe878872f44e2b8088cf0997f2de3e
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A reliable set of reference genes to normalize oxygen-dependent cytoglobin gene expression levels in melanoma

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