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

Joey De Backer,David Hoogewijs,Matthias Bosman,Darko Maric,Sylvia Dewilde

doi:10.1038/s41598-021-90284-6

Joey De Backer, David Hoogewijs + Show 3 more

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https://doi.org/10.1038/s41598-021-90284-6

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Journal: Scientific Reports	Publication Date: May 25, 2021
Citations: 13	License type: open-access

Affiliation: University of Antwerp, University of Fribourg

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.

Highlights

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
Response elements for HIF-1, AP-1, and NFAT have been located within the CYGB promoter, all of which are sensitive to h ypoxia[26], and hypoxia-dependent regulation of CYGB mRNA levels was observed in various cell types and tissues[27,28,29,30]
To investigate the stability of eight of the most commonly used reference genes from different functional classes as recommended by Vandesompele and c olleagues[12] (ACTB, UBC, HMBS, SDHA, HPRT1, TBP, B2M and YHWAZ) within a hypoxic setting we set up an experiment containing two melanoma cell lines expressing high and moderately high endogenous CYGB levels, Malme-3M and A375, incubated under either normoxic or hypoxic conditions for 24 h

Summary

Introduction

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. Reliable detection of hypoxia-inducible mRNA levels by qPCR depends on the critical choice of suitable reference genes for accurate normalization. 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). Our results display that B2M and YWHAZ represent the most optimal reference genes to reliably quantify hypoxia-inducible CYGB expression in melanoma cell lines. The skin is a mildly hypoxic environment and oxygen levels are sufficiently low enough to allow stabilization of the hypoxia-inducible factor α (HIF-α) subunit, thereby increasing the expression of established HIF target genes such as carbonic anhydrase IX (CAIX), glucose transporter-1 (GLUT1) and prolyl hydroxylase domain-2 (PHD2)[18,19,20]. Response elements for HIF-1, AP-1, and NFAT have been located within the CYGB promoter, all of which are sensitive to h ypoxia[26], and hypoxia-dependent regulation of CYGB mRNA levels was observed in various cell types and tissues[27,28,29,30]

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