Abstract
BackgroundReference genes are often interchangeably called housekeeping genes due to 1) the essential cellular functions their proteins provide and 2) their constitutive expression across a range of normal and pathophysiological conditions. However, given the proliferative drive of malignant cells, many reference genes such as beta-actin (ACTB) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) which play critical roles in cell membrane organization and glycolysis, may be dysregulated in tumors versus their corresponding normal controlsMethodsBecause Next Generation Sequencing (NGS) technology has several advantages over hybridization-based technologies, such as independent detection and quantitation of transcription levels, greater sensitivity, and increased dynamic range, we evaluated colorectal cancers (CRC) and their histologically normal tissue counterparts by NGS to evaluate the expression of 21 “classical” reference genes used as normalization standards for PCR based methods. Seventy-nine paired tissue samples of CRC and their patient matched healthy colonic tissues were subjected to NGS analysis of their mRNAs.ResultsWe affirmed that 17 out of 21 classical reference genes had upregulated expression in tumors compared to normal colonic epithelial tissue and dramatically so in some cases. Indeed, tumors were distinguished from normal controls in both unsupervised hierarchical clustering analyses (HCA) and principal component analyses (PCA). We then identified 42 novel potential reference genes with minimal coefficients of variation (CV) across 79 CRC tumor pairs. Though largely consistently expressed across tumors and normal control tissues, a subset of high stage tumors (HSTs) as well as some normal tissue samples (HSNs) located adjacent to these HSTs demonstrated dysregulated expression, thus identifying a subset of tumors with a potentially distinct and aggressive biological profile.ConclusionWhile classical CRC reference genes were found to be differentially expressed between tumors and normal controls, novel reference genes, identified via NGS, were more consistently expressed across malignant and normal colonic tissues. Nonetheless, a subset of HST had profound dysregulation of such genes as did many of the histologically normal tissues adjacent to such HSTs, indicating that the HSTs so distinguished may have unique biological properties and that their histologically normal tissues likely harbor a small population of microscopically undetected but metabolically active tumors.
Highlights
Reference genes are often interchangeably called housekeeping genes due to 1) the essential cellular functions their proteins provide and 2) their constitutive expression across a range of normal and pathophysiological conditions
Confirmation of Next Generation Sequencing (NGS) data accuracy with tumor landmark genes Before investigating the expression of classical reference genes by NGS, we initially examined our 79 colorectal cancers (CRC) cohort for expression levels of known CRC landmark genes including MYC, cyclin dependent kinase 4 (CDK4) and Cyclin D1 (CCND1), as these genes have been shown to be uniformly overexpressed in CRC [21,22,23]
We found that 13 out of 15 genes involved in ribosome biogenesis, a noted hallmark of cancer biology [24], were upregulated in all 79 CRCs (Additional file 1: Figure S1a), with tumors clearly separated from normal tissues in hierarchical clustering analyses (HCA) as well as principal component analyses (PCA) (Additional file 1: Figure S1b, 1c)
Summary
Reference genes are often interchangeably called housekeeping genes due to 1) the essential cellular functions their proteins provide and 2) their constitutive expression across a range of normal and pathophysiological conditions. Given the proliferative drive of malignant cells, many reference genes such as beta-actin (ACTB) and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) which play critical roles in cell membrane organization and glycolysis, may be dysregulated in tumors versus their corresponding normal controls. Basic cellular functions are supported by guaranteed expression of genes encoding proteins mediating important proteins for cellular integrity. Such genes have been referred to as “housekeeping” genes or, for purposes of comparison of gene expression levels across different cell populations, as “reference” genes. While all cells require the functions of proteins encoded by such genes, the uniformity of expression levels in distinct cells and tissues is not confirmed, as diverse physiological conditions and disease states impose different metabolic and structural requirements [1,2,3,4]. We inquired into the presence of no novel reference genes, better suited for quantitative purposes in PCR based assays, based on limited CV across 79 CRC tumor pairs
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