Normalizing to GADPH jeopardises correct quantification of gene expression in ovarian tumours – IPO8 and RPL4 are reliable reference genes

Most cases of ovarian cancer are diagnosed at an advanced stage, with poor prognosis for the patients. Early stages of ovarian cancer are, on the other hand, more accessible to treatment and have much better prognosis. There is an ongoing search for biomarkers with capacity to detect in particular early stages of the disease in screening programs, since this would be the single most important step towards improving the prognosis. A selective biomarker might, furthermore, be helpful in the preoperative assessment of ovarian lesions in order to employ optimal surgery.

Analysis of gene expression by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR), a sensitive technique with broad dynamic range, is a frequent approach for the biomarker discovery in tumour tissue. However, in order to obtain reliable results by RT-qPCR in heterogeneous clinical samples, the expression of a target gene needs to be normalized to a stably expressed reference gene (RG) to minimize the influence of variations in, e.g. extraction yield, reverse-transcription yield, and amplification efficiency [1]. Stability of such reference genes has to be validated in benign and malignant tissues from the specific organ studied. Use of an unstable reference gene will inevitably produce erroneous results. Needless to say, this requirement applies also for ovarian tumours with different differentiation grades and histological types.

The traditionally used house-keeping gene, glyceraldehyde-3-phosphate dehydrogenase (GADPH), was reported to display many diverse activities unrelated to its glycolytic function (e.g. apoptosis and DNA replication) [2], and to be up-regulated in prostate cancer already in the 1990s [3]. The most commonly used RT-qPCR reference genes used for ovarian studies has been GADPH (~40%), β-actin (ACTB) (~20%), ribosomal RNA (18S and 28S rRNA) (~10%) and hypoxanthine phosphoribosyl transferase 1 (HPRT1) (<3%) [4]. More recent study has advised against the use of GADPH and ACTB as RG’s, due to their numerous pseudogenes present in the human genome [5].

Up to now, only two studies have focused on finding a reliable RG in normal ovarian tissue, and benign and malignant serous ovarian tumours. The obtained results, however, differ; Li et al. recommended combination of GUSB, PPIA, and TBP [4], whereas Fu et al. concluded that combination of RPL4, RPLPO, and HSP90AB1 (HSPCB) are more suitable [6]. Both studies were performed on Chinese populations, did not include borderline tumours, and used SYBR Green RT-qPCR technique.

The present study was performed on a Scandinavian population, included borderline tumours, used predesigned commercial RT-qPCR probes, and applied four different statistical software programs. In addition to the above mentioned traditionally used and earlier recommended RGs for ovarian tissue, we also selected four genes from a commercially printed array (ABL1, CDKN1A, IPO8, and RPL30). Thus, altogether 13 genes we included in the study. Finally, two target genes were chosen to demonstrate the divergent results, which may be obtained by normalizing their mRNAs to suitable vs. unsuitable RGs: G protein-coupled estrogen receptor (GPER), which has no differences in expression between benign and malignant ovarian tumours and urokinase plasminogen activator receptor (uPAR), which is up-regulated in malignant tumours.

Although RT-qPCR is the most commonly used method for assessing gene expression, in-depth studies of potential reference genes and their expression pattern in ovarian tumour tissue are insufficient. The aim of this study was to identify the most stably expressed RGs, which can be recommended for normalization of RT-qPCR results in benign, borderline and malignant ovarian tumour samples.

We analysed the traditionally used RGs, those reported as being suitable for ovarian tissue, and the four most promising genes from a commercial RG array. Altogether 13 potential reference genes were tested for stability across groups of benign, borderline, and malignant primary ovarian tumours of different histological subtypes. Of the genes studied, IPO8, RPL4, TBP, RPLPO, and ACTB were found to be the most stable according to the statistical applets GeNorm, NormFinder and BestKeeper. Our findings on RPL4, RPLPO, and TBP in a Scandinavian population are in accordance with previous reports in Asian populations [4, 6]. In contrast, our results did not support PPIA as suitable RG, which has been observed previously [4]. With regard to the heterogeneity of ovarian tumour materials and different ranking results produced by the commonly used statistical approaches, we decided to further employ the Equivalence test in our analysis. By applying strict criteria in the Equivalence test, i.e. only allowing a 2-fold change of expression, we could identify IPO8 expression as the most stable of all candidate genes tested.

We included IPO8 in our study because it showed low variation in expression between the benign and the malignant sample in the commercial array. This gene was equivalently expressed across the tumour subgroups of different malignant potential and histology. IPO8 is a Ran-binding protein mediating nuclear import [15] and has been already reported stably expressed in lung tissues [16], gliomas [17], and colon cancer [18].

The second best RG for group-wise comparison, RPL4, encodes a protein that is a component of the 60S ribosome subunit [19]. Apart from ovarian tissue, it has previously been recommended as RG in combination with PGK1 for exfoliated cervical cells [20]. RPLPO, another gene from the ribosomal protein family, had stable expression in HPV-positive as in HPV-negative cervical samples [21] and in tamoxifen or estrogen treated breast cancer cells [22]. TBP, a key regulator of gene expression, has previously been identified as a suitable RG for expression studies on human hepatitis B virus-related hepatocellular carcinoma [23], human renal cell carcinoma [24], and glioblastomas [17]. RPLPO and TBP also belonged to one of the most stably expressed genes in breast carcinomas [25].

Two other candidates that have not previously been tested as RGs in ovarian tumour tissue, ABL1 and CDKN1A, were selected from the commercial gene array. Both genes satisfied the Equivalence test at 3-fold expression change. ABL1, originally identified as a homologue of the transforming gene of the Abelson murine leukemia virus, is a proto-oncogene, which has been implicated in mitogenesis, regulation of gene transcription, and inhibition of apoptosis [26]. Nucleotide polymorphism in the ABL1 gene has been associated with risk of ovarian cancer [27]. CDKN1A (also known as p21) was initially described as an inhibitor of cancer cell proliferation [27]. However, recent studies suggest that it has dual functions since it also may promote tumour progression [28] and be associated with cisplatin resistance in ovarian cancer [29].

According to BestKeeper and Equivalence test criteria, we found that GADPH had the worst expression stability in our set of ovarian tumour samples. Similar unfavourable results were obtained for HPRT1. These observations are in line with previous studies on other tissue types that have discouraged use of GADPH and HPRT1 as RGs for clinical lung specimens [16] and renal cell cancer [24]. Most recently, a microarray study identified a group of genes highly correlated to GADPH up-regulation in various solid tumours, which were and proportionally associated with advanced stages [30]. Previous reports on GADPH in ovarian tissue have either pointed out higher expression in malignant than in benign tumours and normal tissue [6], or not meeting the GeNorm stability criteria [4]. We further demonstrated that employment of GADPH or HPRT1 for normalization resulted in erroneous conclusions on expression of target genes.

To our knowledge, this is the first report on RGs in ovarian tumours that include borderline tumours in addition to benign and malignant tumours. Since they are considered a non-invasive pre-stage of molecular type I ovarian cancer, it is important to include them in any study on biomarker discovery [31].

Ovarian cancer comprises tumours of different morphology and pathogenesis, which may have different gene expression profiles [32]. Therefore we wished to see whether the histology of ovarian tumours influences the stability of RGs. Thus, in contrast to the previous studies conducted exclusively on serous malignant tumours, our study also included mucinous and endometrioid tumours. However, small number of samples in some groups limited the comparisons that could be performed.

In conclusion, thorough statistical evaluation of our 13 candidate RGs identified IPO8 followed by RPL4 as the most suitable for the normalization of gene expression data in benign, borderline, and malignant ovarian tumours. For the first time, IPO8 is presented as the best normaliser for gene expression studies on ovarian tumour tissue with heterogeneous histology when used as a single RG. Neither GADPH nor HPRT1 should be used as RGs for ovarian tissue studies, because of poor expression stability. Normalizing to these genes may erroneously influence the quantification of the target gene(s) and hence reduce the reliability of the RT-qPCR results.