BACKGROUND
comparison of gene expression levels between individuals and/or biological or pathological conditions requires internal reference such as so-called housekeeping genes , so that normalized expression can correct for variations in amounts of starting rna and/or minimize biases due to reverse transcription efficiency.
the ideal hkg selected for normalization should not be influenced by experimental or biological conditions to be compared; in particular, its own expression level should be identical in tumors and adjacent healthy sites.
frequently, reports focus on a gene of interest  and the reference gene is assumed to remain stable without validation.
however, a growing number of studies showed that the expression levels of many commonly used hkgs are affected by experimental conditions or vary in pathological states, particularly in cancer, and thus stressed the risk of blind use of classical hkgs  <cit> .
for instance, despite wide use, gapdh and beta-actin should no longer be considered suitable as internal references in so diverse conditions such as cell proliferation, differentiation, metabolic changes, hypoxia and cancer  <cit> .
since no universal reference has yet been discovered, the choice of hkg should be validated in each condition studied.
to this aim, bioinformatics tools such as normfinder, bestkeeper and genorm have been recently developed  <cit> .
y-box protein  <dig> or ybx <dig> , also known as csda , zonab -associated nucleic acid binding protein) or dbpa , is a multifaceted epithelial-specific protein able to  act as a transcription factor,  regulate mrna stability and  interact with and regulate function of other proteins.
ybx <dig> is thus implicated in the regulation of epithelial morphogenesis and homeostasis by modulating multiple cellular processes such as the control of cell density, proliferation, differentiation, or survival.
for instance, ybx <dig> interacts with the cell cycle regulating kinase cdk <dig>  and upregulates pcna  and cyclin-d <dig> gene transcription  <cit> .
in kidney proximal tubular epithelial cells, ybx3/zonab/csda not only stimulates proliferation, but also represses genes involved in apical differentiation such as cubilin and megalin/lrp- <dig>   <cit> .
more recently, ybx <dig> binding to the p <dig> mrna was shown to stabilize and enhance p <dig> mrna translation, thereby promoting cell survival in response to cellular stress  <cit> .
other studies have implicated ybx <dig> in tumor development.
although ybx <dig> alone is not sufficient to induce liver tumor development  <cit> , its overexpression and nuclear localization in hepatocarcinomas have been correlated to poorer prognosis  <cit> .
ybx <dig> up-regulation has also been reported to play a role in the pathogenesis of gastric cancer, increasing cell invasion and tumor chemoresistance  <cit> .
in opposition, ybx <dig> would display anti-oncogenic effects in squamous cell carcinomas, inhibiting tumor growth and metastasis  <cit> .
clear cell renal cell carcinomas  rank among ten most frequent cases of cancer death in developed countries because of its bad prognosis, linked to a high propensity to metastasize.
our laboratory recently explored the expression level of ybx <dig> in a limited number of non-graded ccrcc as part of a wide study of ybx3/zonab regulation in vitro and in vivo  <cit> .
using beta-actin as reference, ybx <dig> was found to be overexpressed, although the group of jung demonstrated that beta-actin was not appropriate for ccrcc studies  <cit> .
moreover, the only other study of ybx <dig> expression in ccrcc reported only modest overexpression in only one out of ten tumor cases studied  <cit> .
we thus here re-evaluated the expression level of ybx <dig> as well as other genes implicated in ccrcc in a larger panel of ccrcc samples , each compared to adjacent non tumoral tissue, and evaluated  <dig> different hkgs to select for best reference.
we confirmed by multiple analysis that beta-actin is not appropriate as internal reference for studies of gene expression in ccrcc but found that combination of two hkgs, ppia  and rps <dig> , minimizes fluctuations when comparing ccrcc samples to their adjacent healthy tissues.
based on this combined reference, we found no global difference in ybx <dig> expression in ccrcc when analyzed all together, as compared to normal tissue.
however, stratification of ccrcc samples according to tumor grade revealed that global ybx3/zonab/csda expression level is higher in the low-grade tumors and lower in the high-grade tumors.
methods
samples
the  <dig> ccrcc tumor samples and their matched healthy tissues used in this publication were provided by the ucl biolibrary at the cancer centre of the cliniques universitaires saint-luc , brussels, belgium, project #cdcuclr, approved by the biomedical ethics committee of the université catholique de louvain.
all tissue samples are provided by the biolibrary according to the belgian law, with the informed consent of the patients and full agreement from the biomedical ethics committee of the université catholique de louvain.
samples and data are coded anonymously.
patients’ tumor clinical features are compiled in table  <dig>
*, primary ccrcc of the left kidney 19 years ago, treated by surgery.
other ccrcc of the right kidney and metastasis in the thyroid 7 years ago, treated by surgery.
metastasis in the pancreas 3 years ago, treated by surgery.
lobular carcinoma of the right breast treated by mastectomy, axillary dissection, radiotherapy and tamoxifen 3 years ago.
two experimented senior pathologists  independently checked all tissues for diagnosis confirmation, percentage of tumor cells and tumor grading.
all tumor samples sections were found to be mainly composed of tumor cells ; all “normal” paired samples were tumor-free, with variable fibrosis.
total rna isolation and reverse transcription
several cryostat slices  of oct-embedded frozen tissue were pooled and homogenized in 500 μl of trizol , followed by addition of 30 μg glycogen.
lysates were transferred on phase-lock gel  to improve phase separation and to avoid dna contamination.
100 μl chloroform was added, samples were vigorously shaken then centrifuged at 4°c .
the upper phase was transferred into a fresh tube and rna was precipitated with isopropanol  at -20°c overnight, followed by 15 min centrifugation as above.
rna pellets were washed in 75% ethanol and finally resuspended in 30 μl rnase/dnase-free water.
concentration was determined in a nanodrop spectrophotometer and the quality of rna was controlled by 260/ <dig> and 260/ <dig> absorbance ratios .
for reverse transcription, 1 μg of total rnas in 10 μl rnase/dnase-free water was first denaturated by heating at 85°c for 5 min, followed by rapid cooling on ice.
10 μl containing  <dig> units mmlv reverse transcriptase with buffer, 200 ng of primer random p <dig> oligonucleotides, 500 μm dntp, 10 mm dtt, and  <dig> units of rnase out  were then added to denatured rna.
the mixture was incubated successively at 25°c for 15 min, 37°c for 1 h, and 75°c for 10 min.
finally, cdna was diluted to a final concentration of 10 ng of starting rna per μl and stored at -20°c.
real-time qpcr
rtqpcr experiments were performed in 10 μl containing 1 μl of cdna, 200nm of gene specific primers and 5 μl of 2x kapa sybr fast qpcr master mix .
white  <dig> wells plates  were used on a biorad cfx <dig> thermocycler.
cycling conditions were as follows: an initial step at 95°c 5 min for enzyme activation, followed by 45 cycles alternation of 3 sec at 95°c; 30 sec at 60°c and a final dissociation step.
primers used are listed in table  <dig>  primers for ybx3/csda , cycd <dig>  18s ribosomal rna and beta-actin have been published  <cit> .
primers for pcna were from rtprimerdb database .
for megalin/lrp- <dig> and cubilin we resorted to taqman probes .
the other targets were designed using ncbi primerblast .
only primer pairs located in different exons separated by an intronic sequence of at least 1000 bp were considered.
specificity was verified  in silico, by blast analysis on homo sapiens refseq rna ; and  experimentally, based on a single dissociation peak in melting curve analysis of cdna samples and absence of amplification in negative controls .
amplicon sizes were checked on agarose gel and pcr efficiency was assayed for all primer sets by serial dilutions of cdna and reported in table  <dig> according to rasmussen formula: e = 10-1/slope <cit> .
as all primer sets exhibited a comparable efficiency around  <dig> , the 2-ΔΔct formula, where Δct = ctgoi – cthkg, and ΔΔct = Δcttumor – Δctnormal, was used to calculate the fold-expression in tumours compared to matched normal tissue  <cit> .
when the pair of hkgs  was used to normalize gene expression, cthkg in the above formula was the geometric mean of the two ct values.
to homogenize runs, the pcr threshold used to determine ct was systematically set at 1000rfu, i.e.
at the middle of the logarithmic phase of sybr incorporation.
for each primer set, variability was assessed in  <dig> to  <dig> independent pcr runs; mean variations were inferior to 2% except for 18s, which reached  <dig> %.
2
database source for sequence design is the ncbi gene browser .
pcr efficiency was calculated with standard curves  according to rasmussen formula  <cit> .
.
note that bases indicated in bold text in ybx <dig> primers correspond to mismatches in the ybx <dig> pseudogene  <dig> sequence .
primers design for ybx3
for detection of ybx <dig> mrna, two primer pairs were used.
primers used previously  <cit>  are here called “ybx3 + Ψ” , as two independent in silico pcr programs  genome browser  <cit> .
ncbi primer blast) revealed that these primers co-amplified ybx <dig> mrna but also that of a pseudogene  located on human chromosome  <dig>  accordingly, a new pair of primers specific to the authentic gene, named “ybx3-Ψ”, was designed with primer-blast, using same criteria as above.
we found that the sense “ybx3-Ψ” primer hybridized perfectly to ybx <dig> and ybx3p <dig>  while the antisense harbored four bases that differ from ybx3p <dig>  two at the 3′ end and two at the 5′ end .
specificity was confirmed by in silico analysis; however despite the  <dig> divergent bases in the antisense primer, ybx3-Ψ primers could amplify in vitro the ybx3p <dig> pseudogene from genomic dna.
we tested the efficiency of the two ybx <dig> primer pairs on serial dilutions of genomic dna, from 1 ng to 15 pg .
in that range the amplification efficiency with ybx3-Ψ primers was reduced as compared to the amplification with ybx3 + Ψ primers: 82% vs 96%.
immunohistochemistry
cryostat slices  of oct-embedded frozen tissue were stained with the anti-ybx <dig> antibody  according to the protocol described .
images were acquired using a zeiss mirax midi microscope.
statistics
statistical analyses were performed using graphpad prism .
we selected the non-parametric wilcoxon test for paired data to compare hkg levels and normalized gene expression levels between tumor and healthy tissues.
the non-parametric mannwhitney test was used to compare ybx <dig> expression between graded tumor groups.
correlation coefficients were calculated using spearman rank method.
for all analyses, a p <  <dig>  was considered as statistically significant.
to determine hkg stability, we used the reffinder free online access .
reffinder integrates the currently available major computational programs  to compare and to rank the candidate reference genes.
RESULTS
expression and stability of hkg in ccrcc
to optimize normalization of target genes expression in ccrcc, we measured the expression levels of  <dig> candidate hkgs in a panel of  <dig> ccrcc and paired non-malignant tissue.
we compared levels of the widely used 18s ribosomal rna, beta-actin , glyceraldehyde-3-phosphate dehydrogenase  and beta-2-microglobulin  mrnas, as well as mrnas for the less classical rna polymerase type ii  and peptidylprolyl isomerase a , and the recently described potential hkgs ribosomal protein l <dig>  and ribosomal protein s <dig>   <cit> .
results were first analyzed using standard statistical tools.
figure  <dig> shows raw ct values obtained in all tumors  and normal samples , presented as box and whiskers plots.
as expected, values for 18s ribosomal rna  were lower than mean ct values of the seven other hkgs tested .
since ct values for gapdh, 18s, b2m, and rpl <dig> rnas were significantly different between tumor and normal samples , these should be a priori disqualified to normalize gene expression in ccrcc samples .
in contrast, the mean expression levels of the four other mrnas tested, rp <dig>  β-act, ppia and rps <dig> did not vary significantly between the two groups .
among these, ppia and rps <dig> exhibited the most comparable ct values between tumor and healthy tissues .
the stability of the potential hkg was also studied by calculating the coefficient of variation of raw ct  obtained in each group , and in all the biological samples whatever their diseased state  .
globally, the expression levels of all the hkgs tested were less stable in tumor samples  than in normal samples .
the larger variability of hkg among tumor samples as compared to normal tissues from which they arise underline the fact that each tumor is unique and may exhibit genomic instability.
the coefficient of variation of each hkg in all biological samples taken as a whole  was not dependent on the level of expression or ct values  but correlated to their wilcoxon p value testing statistical difference between normal and tumoral groups.
these variations illustrate the differences that do exist among individuals.
these observations underscore the need to work with matched tissues and to critically select the best hkg in order to minimize biases when assaying gene expression.
statistical differences between ct for tumoral versus normal tissue were assessed by non-parametric paired wilcoxon test.
highest p values indicate best candidates; * indicates unsuitable hkg.
coefficients of variation  are given for normal , tumoral  and all confounded  samples.
lower values reflect lesser dispersion and better stability.
altogether, this standard statistical analysis disclosed that ppia and rps <dig> displayed the most comparable and stable expression levels between normal and tumor samples, thereby best qualified as internal reference in ccrcc samples.
this conclusion, and the respective values of the other rnas used for normalization, were further tested using dedicated algorithms available online.
reffinder  is a user-friendly web-based comprehensive tool developed to evaluate and screen reference genes from large experimental datasets.
it integrates the currently available major computational programs, genorm, normfinder, bestkeeper, and the comparative Δct method, to compare and rank the candidate reference genes according to their dispersion as a stability score  <cit> .
a gene with a low score has a stable expression and will thus be better ranked.
based on ranking in each of these programs, reffinder assigns an appropriate score to each individual hkg and calculates the geometric mean of their scores in a final ranking.
as this in silico tool only considers stability of gene levels, but not different biological populations, it is comparable to the ctcv analysis .
thus, when comparing several experimental conditions  it should be used in combination with inter-group statistical analysis as above.
table  <dig> summarizes rankings obtained with the different algorithms for the eight hkgs.
this in silico analysis on the entire  cohort of samples confirmed that ppia and rps <dig> mrnas belong to the most stable housekeeping gene product tested.
interestingly, beta-actin mrna was less stable: except with bestkeeper, it was scored in the second half of the four other rankings.
the genorm algorithm not only supported best rankings of ppia and rps <dig> evidenced with the other algorithms, but also provides search for best combinations of hkgs, which, not surprisingly, was ppia with rps <dig> .
although rpl <dig> could also be considered as a good hkg in terms of stability based on this program, with an identical stability score as ppia , paired analysis revealed that its level varied statistically between tumors and healthy tissues .
each hkg  is ranked according to the indicated algorithms  and reffinder assigned a final rank .
lower values indicate more stable genes.
note that  ppia and rps <dig> rank at the top of  <dig> out of  <dig> algorithms;  gapdh and b2m are worst candidates; and  beta-actin ranks intermediate.
these results and analysis of candidate hkgs in ccrcc thus supported and extended the study of jung  <cit> , which already warned against using beta-actin mrna as only internal reference to normalize rt-qpcr results in ccrcc, and proposed ppia as alternative.
our analysis identified rps <dig> as an alternative good hkg for ccrcc.
interestingly, the genorm algorithm indicated that rps <dig> and ppia together provide the best combination of tested hkgs for gene expression studies in ccrcc.
we propose that both approaches  could be advantageously used to identify the most reliable hkg or combination thereof.
in the second part of this study, we thus used the objectively selected combination of ppia and rps <dig> as reference genes to normalize that of other genes in ccrcc samples.
critical evaluation of ybx3/zonab/csda expression in ccrcc
using this selected combination of hkg mrnas, we thus re-evaluated the expression of ybx <dig> in  <dig> ccrcc tumor samples and paired normal tissue, including the  <dig> samples of our previous study for which normal tissue was available  <cit> .
ybx <dig> gene  is located on chromosome  <dig> at locus p <dig>  and encodes two spliced mrna isoforms .
however, human genome also contains a pseudogene for ybx <dig>  ybx3p <dig>  on chromosome  <dig>  <cit>  .
this intron-less sequence is almost identical to ybx <dig> transcript  and could introduce a bias in mrna quantification through genomic dna contamination, but also if this pseudogene is transcriptionally active  <cit> .
to avoid this possibility, we designed a second pair of primers aimed at distinguishing cdna derived from authentic ybx <dig> mrna vs its pseudogene.
the first pair, used previously  <cit> , was noted ybx3 + Ψ, because two independent in silico pcr programs  showed that these co-amplify ybx <dig> mrna and its pseudogene.
the second pair of primers, named ybx3-Ψ, was designed to be specific for authentic ybx <dig> transcript only.
although the same in silico pcr analysis confirmed its specificity, in vitro experiments on genomic dna revealed that ybx3-Ψ pair could amplify the pseudogene from genomic dna .
however, at low - or real - contaminating genomic dna concentration, ybx3-Ψ primers amplified the pseudogene with a reduced efficiency as compared to ybx3 + Ψ. we thus preferred  the ybx3-Ψ primers pair.
figure  <dig> shows ybx <dig> expression as dot plot.
expression of ybx3+/- Ψ in all  <dig> pairs of samples was first normalized to beta-actin  in order to compare with results from our previous study  <cit> , then to the geometric mean of the optimal combination of selected hkgs for ccrcc identified above, ppia and rps <dig> .
the relative ybx <dig> mrna expression in tumors was presented as ratio to the corresponding value in adjacent non-tumoral tissue, set as the unity.
in most cases, there was little difference in ybx <dig> quantification when comparing the two pairs of primers .
when expression of ybx <dig> was normalized to that of β-act, global analysis of the  <dig> samples revealed a wider distribution and a significant  <dig> -fold ybx <dig> overexpression in tumors, yet limited to about half samples and at a modest, if not marginal level.
of the three samples used in the previous study  <cit> , sample # <dig>  and # <dig>  showed the highest apparent overexpression of ybx <dig> when normalized to beta-actin.
however, when normalized to the geometric mean of ppia and rps <dig>  these samples display a  <dig> - and  <dig> -fold expression.
this illustrates the strong impact of the choice of hkg to normalize gene expression, and the need to carefully validate this internal reference a priori.
moreover, it emphasizes the need to re-evaluate ybx <dig> expression in ccrcc.
when the geometric mean of ppia and rps <dig> expression levels was used to normalize authentic ybx <dig> expression in the  <dig> samples, the distribution was less dispersed and this cohort no longer showed a significant difference in ybx <dig> expression in tumor samples, as compared to matched controls , in agreement with the report by kohno and colleagues  <cit> .
expression of other ccrcc-associated genes
to ensure that the combination of hkgs selected for normalization of gene expression in ccrcc did not introduce an opposite bias , we further analyzed the expression of key genes known to be implicated in the pathogenesis of, and overexpressed in, ccrcc: vegfa, cmyc and cyclin-d <dig>
the most prevalent cause of ccrcc  is inactivation of von hippel-lindau  gene, by allelic deletions, mutations, or epigenetic silencing  <cit> .
inactivation of vhl results into stabilization of hypoxia inducible factors  and their accumulation in the nucleus, thereby inducing a panel of genes including vegfa , pdgf , egf , and tgf  that ultimately lead to neo-angiogenesis and tumor progression  <cit> .
blocking the vegf pathway indeed emerged as a promising therapeutic strategy in ccrcc  <cit> .
even if less studied than the vhl/hif/vegfa pathway, the c-myc proto-oncogene and cyclind <dig> have also been clearly implicated in kidney tumorigenesis.
as is the case in many other cancers, c-myc is overexpressed in a majority of clear cell renal tumors  <cit> , sometimes associated with c-myc gene locus amplification  <cit> , and is essential for enhanced proliferation of ccrcc tumor cells  <cit> .
the pro-proliferative cyclind <dig> is a c-myc target and is highly expressed in 50-75% ccrcc cases  <cit> .
moreover, the vhl pathway has been reported to control c-myc  <cit>  and cyclind <dig>  <cit> .we thus measured the expression of vegfa, c-myc and cyclind <dig> in the  <dig> tissue pairs and normalized their expression to that of the geometric mean of ppia and rps <dig> .
tumor samples clearly exhibited a significant overexpression of vegfa , c-myc  and cyclind <dig> , as compared to their matched normal tissues .
normalization with the combined references genes thus confirmed published data, indicating they did not introduce masking bias.
expression of ybx <dig> target genes
we then assessed the expression of known targets of ybx3/csda/zonab in the ccrcc samples and studied the correlation of their expression with that of ybx <dig>  cyclind <dig> and proliferative cell nuclear antigen  contain ybx3-responsive elements in their promoters and are stimulated by ybx <dig> .
on the contrary, megalin and cubilin genes also contain “ccaat” boxes in their regulatory regions, but in these cases ybx <dig> binding represses transcription .
in the  <dig> tumor tissues, cyclind <dig> expression was stimulated as compared to their paired samples .
interestingly, variations of cyclind <dig> expression in the cohort correlated with ybx <dig> expression : low ybx3-expressing tumors had low levels of cyclind <dig>  while high ybx <dig> tumors displayed high cyclind <dig> mrnas.
pcna expression level in the ccrcc tumors was not different from normal tissues and accordingly no correlation was found with ybx <dig> .
cubilin, which is repressed by ybx <dig> in vitro, was discarded from our analysis as we could not reproducibly amplify cubilin mrna in all the  <dig> paired samples.
megalin/lrp- <dig> has been shown by microarray and rt-qpcr to be overexpressed in clear cell as well as in papillary rcc  <cit> .
this observation was confirmed in our  <dig> tumor samples that exhibited a significant overexpression of megalin/lrp- <dig> as compared to their paired controls .
correlation of megalin expression with ybx <dig> showed a trend to an inverse relationship but did not reach statistical significance .
this correlation study suggests that in ccrcc, ybx <dig> level of expression may affect cyclind <dig> expression and may also participate in megalin/lrp- <dig> expression.
finally, we re-analysed beta-actin, not only as a classical hkg, but also as a gene per se based on its normalized expression by reference to combined ppia and rps <dig>  as already observed in figure  <dig>  β-act expression in the  <dig> tumor tissues was variable and exhibited a trend to be globally down-regulated in ccrcc samples, even if not reaching statistical significance .
this observation again argues against the use of beta-actin as a reference when measuring gene expression in ccrcc, and requires one to reconsider ybx <dig> overexpression in most cases of our previous study  <cit> .
intriguingly, some reports suggest a possible role of actin expression in the tumorigenesis process.
actin is a major component of cytoskeleton, and altered cell morphology is a characteristic of tumor cells, notably to enable invasiveness, migration and metastasis.
for example, accumulation of beta-actin in tips of pseudopodia drives invasiveness and metastatic ability of transformed mdck cells  <cit> .
indeed, even though mechanisms are not yet well understood, changes in expression level of beta-actin were associated with higher invasiveness of hepatoma cells  <cit> , and with metastatic potential of colon adenocarcinoma cell lines  <cit> .
besides its direct role in the control of cell morphology, actin dynamics could also regulate genetic programs by the so-called mechanogenetic mechanisms  <cit> .
indeed, ablation of beta-actin altered the ratio of globular actin to filamentous actin in mouse embryonic fibroblasts, with corresponding changes in expression of genes regulating cell cycle and motility  <cit> .
interestingly, except for one obvious outlier, variations of beta-actin expression in the cohort we analyzed appeared to correlate with ybx <dig> expression .
variations of beta-actin expression also followed in parallel ybx <dig> expression during mouse kidney embryonic development .
based on the well-known roles of ybx <dig> in the control of epithelium morphogenesis, one can hypothesize that ybx <dig> could regulate beta-actin expression during kidney ontogenesis and oncogenesis.
expression of ybx3/zonab/csda as a function of tumor grade
although we concluded above that ybx <dig> did not show systematic overexpression in the global cohort of ccrcc samples, a closer look suggested the possibility of three expression groups: high, intermediate, and low.
indeed, as compared to matched normal tissues, five tumors  exhibited a significant overexpression of ybx3; five other  exhibited significant underexpression; while the six remaining  did not show significant difference from their normal counterparts .
we thus wondered if these variations in ybx <dig> levels could be related to ccrcc pathogenesis and more specifically to tumor grades and asked two experimented pathologists to independently grade the  <dig> tumor samples according to fuhrman , being totally unaware of our expression level data.
both pathologists agreed on the  <dig> cases; one case was nevertheless excluded from further analysis as it contained 40% of normal parenchyma .
of the  <dig> remaining cases  <dig> tumor samples were graded # <dig>   <dig> were graded # <dig> and  <dig> were graded # <dig>  segmentation of ybx <dig> expression levels according to histopathological grading is shown in figure  <dig>  this analysis revealed that ybx <dig> negatively correlates to tumor grade with higher expression levels in grade # <dig>  intermediate in grade # <dig>  and lower in grade # <dig> .
this correlation was further analyzed at the protein level by immunohistology in tumor samples from grade # <dig> to # <dig> .
ybx <dig> staining was clearly evident in all nuclei of grade # <dig> tumors.
in grade # <dig> tumors, ybx <dig> staining was weaker in the nuclei and sometimes appeared in the cytoplasm.
in grade # <dig> tumors, we observed strong inter- and intra-tumor heterogeneity of ybx <dig> staining; varying from complete absence to intense staining in some nuclei.
these qualitative histopathological observations are in agreement with our rt-qpcr analyses showing decreased expression level of ybx <dig> from grade # <dig> to grade # <dig> and  <dig> .
even if these numbers were small, the negative correlation of ybx <dig> mrna expression level with tumor anaplasia, hence potential progression, was clearly significant.
upregulation of ybx <dig> in grade # <dig> tumors, followed by progressive silencing in more anaplastic, hence more prone to metastasize, could indicate that ybx <dig> plays a biological role at a low degree of neoplastic transformation characterizing tumors with low metastatic potential, and be dispensable for disease progression.
a similar trend has been recently reported in hepatocellular carcinoma .
although ybx <dig> overexpression and nuclear localization was initially correlated to poor patients outcome  <cit> , a recent study by the same team revealed that high ybx <dig> level in pre-cancerous “hypercarcinogenic” states, but not in established hcc, was, in fact, associated to poor prognosis  <cit> .
it is thus conceivable that high ybx <dig> expression would promote dedifferentiation and proliferation, thus secondarily favor mutations of cancer as a multistep process  <cit> .
an interesting alternative hypothesis is based on the ability of ybx <dig> to reduce tumor angiogenesis and lymphangiogenesis, so as to inhibit tumor growth and metastasis in lung cancer and squamous cell carcinoma models  <cit> .
the authors went so far as to propose ybx <dig> as a potential “therapeutic” gene, which, if artificially induced in tumors, could prevent disease progression and dissemination.
of note, ybx <dig> is also down-regulated in breast cancer, as compared to healthy tissue  <cit> .
however, the grade of the breast cancer was not mentioned.
if this interpretation is correct, ybx <dig> extinction in most anaplastic, hence potentially invasive tumor cells, would promote angio/lympho-genesis, thus favor metastatic dissemination.
CONCLUSIONS
in conclusion, although the pathogenic implications of ybx <dig> in  human tumorigenesis clearly need to be further explored, its global mrna expression level may be part of a “signature” of rcc grading.
competing interests
the authors declare that no conflict of interest exists.
authors’ contributions
sd and asd conceived, designed, performed the experiments, analyzed results and drafted the manuscript.
em and j-pc graded tumor samples and helped to draft the manuscript.
pjc analyzed results and drafted the manuscript.
cep supervised the study, analyzed the results and drafted the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
ybx <dig> correlation with known target genes and beta-actin.
dot plots with respective mean fold variations of ybx3-Ψ  and cycld <dig> , pcna , megalin/lrp- <dig>  and b-act   in the  <dig> tumor samples compared to adjacent healthy tissues.
red dotted lines indicate values in adjacent healthy tissues.
black lines show linear regression curves.
please note the logarithmic scale for megalin expression.
spearman coefficient of correlation and p value are indicated.
note that value of sample <dig>  can be considered as outlier  and was excluded.
click here for file
additional file 2
effects of primer and choice of reference hkgs on the estimation of normalized ybx <dig> expression in ccrcc samples.
expression of ybx <dig> mrna, measured with ybx3 + Ψ  and ybx3-Ψ  primers, in each of the  <dig> tumors compared to adjacent healthy tissue  after normalization to either beta-actin , or ppia & rps <dig> .
values are means of  <dig> independent assays with standard deviations.
tumor values statistically different from their normal paired tissues are indicated by asterisks .
pairs  <dig> to  <dig> were those analyzed in our previous report  <cit> .
click here for file
additional file 3
distribution of ybx3 + Ψ expression ratios in tumor samples according to histological grading.
circles are values for ybx3 + Ψ expression ratios in the  <dig> fuhrman-graded tumors compared to paired adjacent healthy tissue after normalization to geometric mean of ppia + rps <dig>  lines are medians for each group and statistical differences between subgroups are indicated as well .
the expression of ybx <dig> inversely correlates with tumor grade.
click here for file
acknowledgements
the authors want to thank the biolibrary at the cancer centre of the cliniques universitaires saint-luc for their help in providing tissue samples and associated patients information.
the work was supported by grants from the université catholique de louvain  and the fund for scientific medical research .
a.-s.d.
is a doctoral fellow from télévie, sd is a post-doctoral fellow, and cep a research associate of the f.r.s.-fnrs .

 comparison of gene expression levels between individuals and/or biological or pathological conditions requires internal reference such as so-called housekeeping genes, so that normalized expression can correct for variations in amounts of starting rna and/or minimize biases due to reverse transcription efficiency. the ideal hkg selected for normalization should not be influenced by experimental or biological conditions to be compared; in particular, its own expression level should be identical in tumors and adjacent healthy sites. frequently, reports focus on a gene of interest and the reference gene is assumed to remain stable without validation. however, a growing number of studies showed that the expression levels of many commonly used hkgs are affected by experimental conditions or vary in pathological states, particularly in cancer, and thus stressed the risk of blind use of classical hkgs. for instance, despite wide use, gapdh and beta-actin should no longer be considered suitable as internal references in so diverse conditions such as cell proliferation, differentiation, metabolic changes, hypoxia and cancer. since no universal reference has yet been discovered, the choice of hkg should be validated in each condition studied. to this aim, bioinformatics tools such as normfinder, bestkeeper and genorm have been recently developed. y-box protein or ybx, also known as csda, zonab -associated nucleic acid binding protein) or dbpa, is a multifaceted epithelial-specific protein able to act as a transcription factor, regulate mrna stability and interact with and regulate function of other proteins. ybx is thus implicated in the regulation of epithelial morphogenesis and homeostasis by modulating multiple cellular processes such as the control of cell density, proliferation, differentiation, or survival. for instance, ybx interacts with the cell cycle regulating kinase cdk and upregulates pcna and cyclin-d gene transcription. in kidney proximal tubular epithelial cells, ybx3/zonab/csda not only stimulates proliferation, but also represses genes involved in apical differentiation such as cubilin and megalin/lrp-. more recently, ybx binding to the p mrna was shown to stabilize and enhance p mrna translation, thereby promoting cell survival in response to cellular stress. other studies have implicated ybx in tumor development. although ybx alone is not sufficient to induce liver tumor development, its overexpression and nuclear localization in hepatocarcinomas have been correlated to poorer prognosis. ybx up-regulation has also been reported to play a role in the pathogenesis of gastric cancer, increasing cell invasion and tumor chemoresistance. in opposition, ybx would display anti-oncogenic effects in squamous cell carcinomas, inhibiting tumor growth and metastasis. clear cell renal cell carcinomas rank among ten most frequent cases of cancer death in developed countries because of its bad prognosis, linked to a high propensity to metastasize. our laboratory recently explored the expression level of ybx in a limited number of non-graded ccrcc as part of a wide study of ybx3/zonab regulation in vitro and in vivo. using beta-actin as reference, ybx was found to be overexpressed, although the group of jung demonstrated that beta-actin was not appropriate for ccrcc studies. moreover, the only other study of ybx expression in ccrcc reported only modest overexpression in only one out of ten tumor cases studied. we thus here re-evaluated the expression level of ybx as well as other genes implicated in ccrcc in a larger panel of ccrcc samples, each compared to adjacent non tumoral tissue, and evaluated different hkgs to select for best reference. we confirmed by multiple analysis that beta-actin is not appropriate as internal reference for studies of gene expression in ccrcc but found that combination of two hkgs, ppia and rps, minimizes fluctuations when comparing ccrcc samples to their adjacent healthy tissues. based on this combined reference, we found no global difference in ybx expression in ccrcc when analyzed all together, as compared to normal tissue. however, stratification of ccrcc samples according to tumor grade revealed that global ybx3/zonab/csda expression level is higher in the low-grade tumors and lower in the high-grade tumors. methods samples the ccrcc tumor samples and their matched healthy tissues used in this publication were provided by the ucl biolibrary at the cancer centre of the cliniques universitaires saint-luc, brussels, belgium, project #cdcuclr, approved by the biomedical ethics committee of the université catholique de louvain. all tissue samples are provided by the biolibrary according to the belgian law, with the informed consent of the patients and full agreement from the biomedical ethics committee of the université catholique de louvain. samples and data are coded anonymously. patients’ tumor clinical features are compiled in table  *, primary ccrcc of the left kidney 19 years ago, treated by surgery. other ccrcc of the right kidney and metastasis in the thyroid 7 years ago, treated by surgery. metastasis in the pancreas 3 years ago, treated by surgery. lobular carcinoma of the right breast treated by mastectomy, axillary dissection, radiotherapy and tamoxifen 3 years ago. two experimented senior pathologists independently checked all tissues for diagnosis confirmation, percentage of tumor cells and tumor grading. all tumor samples sections were found to be mainly composed of tumor cells; all “normal” paired samples were tumor-free, with variable fibrosis. total rna isolation and reverse transcription several cryostat slices of oct-embedded frozen tissue were pooled and homogenized in 500 μl of trizol, followed by addition of 30 μg glycogen. lysates were transferred on phase-lock gel to improve phase separation and to avoid dna contamination. 100 μl chloroform was added, samples were vigorously shaken then centrifuged at 4°c. the upper phase was transferred into a fresh tube and rna was precipitated with isopropanol at -20°c overnight, followed by 15 min centrifugation as above. rna pellets were washed in 75% ethanol and finally resuspended in 30 μl rnase/dnase-free water. concentration was determined in a nanodrop spectrophotometer and the quality of rna was controlled by 260/ and 260/ absorbance ratios. for reverse transcription, 1 μg of total rnas in 10 μl rnase/dnase-free water was first denaturated by heating at 85°c for 5 min, followed by rapid cooling on ice. 10 μl containing units mmlv reverse transcriptase with buffer, 200 ng of primer random p oligonucleotides, 500 μm dntp, 10 mm dtt, and units of rnase out were then added to denatured rna. the mixture was incubated successively at 25°c for 15 min, 37°c for 1 h, and 75°c for 10 min. finally, cdna was diluted to a final concentration of 10 ng of starting rna per μl and stored at -20°c. real-time qpcr rtqpcr experiments were performed in 10 μl containing 1 μl of cdna, 200nm of gene specific primers and 5 μl of 2x kapa sybr fast qpcr master mix. white wells plates were used on a biorad cfx thermocycler. cycling conditions were as follows: an initial step at 95°c 5 min for enzyme activation, followed by 45 cycles alternation of 3 sec at 95°c; 30 sec at 60°c and a final dissociation step. primers used are listed in table  primers for ybx3/csda, cycd 18s ribosomal rna and beta-actin have been published. primers for pcna were from rtprimerdb database. for megalin/lrp- and cubilin we resorted to taqman probes. the other targets were designed using ncbi primerblast. only primer pairs located in different exons separated by an intronic sequence of at least 1000 bp were considered. specificity was verified in silico, by blast analysis on homo sapiens refseq rna; and experimentally, based on a single dissociation peak in melting curve analysis of cdna samples and absence of amplification in negative controls. amplicon sizes were checked on agarose gel and pcr efficiency was assayed for all primer sets by serial dilutions of cdna and reported in table  according to rasmussen formula: e = 10-1/slope. as all primer sets exhibited a comparable efficiency around, the 2-ΔΔct formula, where Δct = ctgoi – cthkg, and ΔΔct = Δcttumor – Δctnormal, was used to calculate the fold-expression in tumours compared to matched normal tissue. when the pair of hkgs was used to normalize gene expression, cthkg in the above formula was the geometric mean of the two ct values. to homogenize runs, the pcr threshold used to determine ct was systematically set at 1000rfu, i.e. at the middle of the logarithmic phase of sybr incorporation. for each primer set, variability was assessed in to independent pcr runs; mean variations were inferior to 2% except for 18s, which reached %. 2 database source for sequence design is the ncbi gene browser. pcr efficiency was calculated with standard curves according to rasmussen formula.. note that bases indicated in bold text in ybx primers correspond to mismatches in the ybx pseudogene sequence. primers design for ybx3 for detection of ybx mrna, two primer pairs were used. primers used previously are here called “ybx3 + Ψ”, as two independent in silico pcr programs genome browser. ncbi primer blast) revealed that these primers co-amplified ybx mrna but also that of a pseudogene located on human chromosome accordingly, a new pair of primers specific to the authentic gene, named “ybx3-Ψ”, was designed with primer-blast, using same criteria as above. we found that the sense “ybx3-Ψ” primer hybridized perfectly to ybx and ybx3p while the antisense harbored four bases that differ from ybx3p two at the 3′ end and two at the 5′ end. specificity was confirmed by in silico analysis; however despite the divergent bases in the antisense primer, ybx3-Ψ primers could amplify in vitro the ybx3p pseudogene from genomic dna. we tested the efficiency of the two ybx primer pairs on serial dilutions of genomic dna, from 1 ng to 15 pg. in that range the amplification efficiency with ybx3-Ψ primers was reduced as compared to the amplification with ybx3 + Ψ primers: 82% vs 96%. immunohistochemistry cryostat slices of oct-embedded frozen tissue were stained with the anti-ybx antibody according to the protocol described. images were acquired using a zeiss mirax midi microscope. statistics statistical analyses were performed using graphpad prism. we selected the non-parametric wilcoxon test for paired data to compare hkg levels and normalized gene expression levels between tumor and healthy tissues. the non-parametric mannwhitney test was used to compare ybx expression between graded tumor groups. correlation coefficients were calculated using spearman rank method. for all analyses, a p <  was considered as statistically significant. to determine hkg stability, we used the reffinder free online access. reffinder integrates the currently available major computational programs to compare and to rank the candidate reference genes.  expression and stability of hkg in ccrcc to optimize normalization of target genes expression in ccrcc, we measured the expression levels of candidate hkgs in a panel of ccrcc and paired non-malignant tissue. we compared levels of the widely used 18s ribosomal rna, beta-actin, glyceraldehyde-3-phosphate dehydrogenase and beta-2-microglobulin mrnas, as well as mrnas for the less classical rna polymerase type ii and peptidylprolyl isomerase a, and the recently described potential hkgs ribosomal protein l and ribosomal protein s. results were first analyzed using standard statistical tools. figure  shows raw ct values obtained in all tumors and normal samples, presented as box and whiskers plots. as expected, values for 18s ribosomal rna were lower than mean ct values of the seven other hkgs tested. since ct values for gapdh, 18s, b2m, and rpl rnas were significantly different between tumor and normal samples, these should be a priori disqualified to normalize gene expression in ccrcc samples. in contrast, the mean expression levels of the four other mrnas tested, rp β-act, ppia and rps did not vary significantly between the two groups. among these, ppia and rps exhibited the most comparable ct values between tumor and healthy tissues. the stability of the potential hkg was also studied by calculating the coefficient of variation of raw ct obtained in each group, and in all the biological samples whatever their diseased state. globally, the expression levels of all the hkgs tested were less stable in tumor samples than in normal samples. the larger variability of hkg among tumor samples as compared to normal tissues from which they arise underline the fact that each tumor is unique and may exhibit genomic instability. the coefficient of variation of each hkg in all biological samples taken as a whole was not dependent on the level of expression or ct values but correlated to their wilcoxon p value testing statistical difference between normal and tumoral groups. these variations illustrate the differences that do exist among individuals. these observations underscore the need to work with matched tissues and to critically select the best hkg in order to minimize biases when assaying gene expression. statistical differences between ct for tumoral versus normal tissue were assessed by non-parametric paired wilcoxon test. highest p values indicate best candidates; * indicates unsuitable hkg. coefficients of variation are given for normal, tumoral and all confounded samples. lower values reflect lesser dispersion and better stability. altogether, this standard statistical analysis disclosed that ppia and rps displayed the most comparable and stable expression levels between normal and tumor samples, thereby best qualified as internal reference in ccrcc samples. this conclusion, and the respective values of the other rnas used for normalization, were further tested using dedicated algorithms available online. reffinder is a user-friendly web-based comprehensive tool developed to evaluate and screen reference genes from large experimental datasets. it integrates the currently available major computational programs, genorm, normfinder, bestkeeper, and the comparative Δct method, to compare and rank the candidate reference genes according to their dispersion as a stability score. a gene with a low score has a stable expression and will thus be better ranked. based on ranking in each of these programs, reffinder assigns an appropriate score to each individual hkg and calculates the geometric mean of their scores in a final ranking. as this in silico tool only considers stability of gene levels, but not different biological populations, it is comparable to the ctcv analysis. thus, when comparing several experimental conditions it should be used in combination with inter-group statistical analysis as above. table  summarizes rankings obtained with the different algorithms for the eight hkgs. this in silico analysis on the entire cohort of samples confirmed that ppia and rps mrnas belong to the most stable housekeeping gene product tested. interestingly, beta-actin mrna was less stable: except with bestkeeper, it was scored in the second half of the four other rankings. the genorm algorithm not only supported best rankings of ppia and rps evidenced with the other algorithms, but also provides search for best combinations of hkgs, which, not surprisingly, was ppia with rps. although rpl could also be considered as a good hkg in terms of stability based on this program, with an identical stability score as ppia, paired analysis revealed that its level varied statistically between tumors and healthy tissues. each hkg is ranked according to the indicated algorithms and reffinder assigned a final rank. lower values indicate more stable genes. note that ppia and rps rank at the top of out of algorithms; gapdh and b2m are worst candidates; and beta-actin ranks intermediate. these results and analysis of candidate hkgs in ccrcc thus supported and extended the study of jung, which already warned against using beta-actin mrna as only internal reference to normalize rt-qpcr results in ccrcc, and proposed ppia as alternative. our analysis identified rps as an alternative good hkg for ccrcc. interestingly, the genorm algorithm indicated that rps and ppia together provide the best combination of tested hkgs for gene expression studies in ccrcc. we propose that both approaches could be advantageously used to identify the most reliable hkg or combination thereof. in the second part of this study, we thus used the objectively selected combination of ppia and rps as reference genes to normalize that of other genes in ccrcc samples. critical evaluation of ybx3/zonab/csda expression in ccrcc using this selected combination of hkg mrnas, we thus re-evaluated the expression of ybx in ccrcc tumor samples and paired normal tissue, including the samples of our previous study for which normal tissue was available. ybx gene is located on chromosome at locus p and encodes two spliced mrna isoforms. however, human genome also contains a pseudogene for ybx ybx3p on chromosome. this intron-less sequence is almost identical to ybx transcript and could introduce a bias in mrna quantification through genomic dna contamination, but also if this pseudogene is transcriptionally active. to avoid this possibility, we designed a second pair of primers aimed at distinguishing cdna derived from authentic ybx mrna vs its pseudogene. the first pair, used previously, was noted ybx3 + Ψ, because two independent in silico pcr programs showed that these co-amplify ybx mrna and its pseudogene. the second pair of primers, named ybx3-Ψ, was designed to be specific for authentic ybx transcript only. although the same in silico pcr analysis confirmed its specificity, in vitro experiments on genomic dna revealed that ybx3-Ψ pair could amplify the pseudogene from genomic dna. however, at low - or real - contaminating genomic dna concentration, ybx3-Ψ primers amplified the pseudogene with a reduced efficiency as compared to ybx3 + Ψ. we thus preferred the ybx3-Ψ primers pair. figure  shows ybx expression as dot plot. expression of ybx3+/- Ψ in all pairs of samples was first normalized to beta-actin in order to compare with results from our previous study, then to the geometric mean of the optimal combination of selected hkgs for ccrcc identified above, ppia and rps. the relative ybx mrna expression in tumors was presented as ratio to the corresponding value in adjacent non-tumoral tissue, set as the unity. in most cases, there was little difference in ybx quantification when comparing the two pairs of primers. when expression of ybx was normalized to that of β-act, global analysis of the samples revealed a wider distribution and a significant -fold ybx overexpression in tumors, yet limited to about half samples and at a modest, if not marginal level. of the three samples used in the previous study, sample # and # showed the highest apparent overexpression of ybx when normalized to beta-actin. however, when normalized to the geometric mean of ppia and rps these samples display a - and -fold expression. this illustrates the strong impact of the choice of hkg to normalize gene expression, and the need to carefully validate this internal reference a priori. moreover, it emphasizes the need to re-evaluate ybx expression in ccrcc. when the geometric mean of ppia and rps expression levels was used to normalize authentic ybx expression in the samples, the distribution was less dispersed and this cohort no longer showed a significant difference in ybx expression in tumor samples, as compared to matched controls, in agreement with the report by kohno and colleagues. expression of other ccrcc-associated genes to ensure that the combination of hkgs selected for normalization of gene expression in ccrcc did not introduce an opposite bias, we further analyzed the expression of key genes known to be implicated in the pathogenesis of, and overexpressed in, ccrcc: vegfa, cmyc and cyclin-d the most prevalent cause of ccrcc is inactivation of von hippel-lindau gene, by allelic deletions, mutations, or epigenetic silencing. inactivation of vhl results into stabilization of hypoxia inducible factors and their accumulation in the nucleus, thereby inducing a panel of genes including vegfa, pdgf, egf, and tgf that ultimately lead to neo-angiogenesis and tumor progression. blocking the vegf pathway indeed emerged as a promising therapeutic strategy in ccrcc. even if less studied than the vhl/hif/vegfa pathway, the c-myc proto-oncogene and cyclind have also been clearly implicated in kidney tumorigenesis. as is the case in many other cancers, c-myc is overexpressed in a majority of clear cell renal tumors, sometimes associated with c-myc gene locus amplification, and is essential for enhanced proliferation of ccrcc tumor cells. the pro-proliferative cyclind is a c-myc target and is highly expressed in 50-75% ccrcc cases. moreover, the vhl pathway has been reported to control c-myc and cyclind.we thus measured the expression of vegfa, c-myc and cyclind in the tissue pairs and normalized their expression to that of the geometric mean of ppia and rps. tumor samples clearly exhibited a significant overexpression of vegfa, c-myc and cyclind, as compared to their matched normal tissues. normalization with the combined references genes thus confirmed published data, indicating they did not introduce masking bias. expression of ybx target genes we then assessed the expression of known targets of ybx3/csda/zonab in the ccrcc samples and studied the correlation of their expression with that of ybx cyclind and proliferative cell nuclear antigen contain ybx3-responsive elements in their promoters and are stimulated by ybx. on the contrary, megalin and cubilin genes also contain “ccaat” boxes in their regulatory regions, but in these cases ybx binding represses transcription. in the tumor tissues, cyclind expression was stimulated as compared to their paired samples. interestingly, variations of cyclind expression in the cohort correlated with ybx expression: low ybx3-expressing tumors had low levels of cyclind while high ybx tumors displayed high cyclind mrnas. pcna expression level in the ccrcc tumors was not different from normal tissues and accordingly no correlation was found with ybx. cubilin, which is repressed by ybx in vitro, was discarded from our analysis as we could not reproducibly amplify cubilin mrna in all the paired samples. megalin/lrp- has been shown by microarray and rt-qpcr to be overexpressed in clear cell as well as in papillary rcc. this observation was confirmed in our tumor samples that exhibited a significant overexpression of megalin/lrp- as compared to their paired controls. correlation of megalin expression with ybx showed a trend to an inverse relationship but did not reach statistical significance. this correlation study suggests that in ccrcc, ybx level of expression may affect cyclind expression and may also participate in megalin/lrp- expression. finally, we re-analysed beta-actin, not only as a classical hkg, but also as a gene per se based on its normalized expression by reference to combined ppia and rps as already observed in figure  β-act expression in the tumor tissues was variable and exhibited a trend to be globally down-regulated in ccrcc samples, even if not reaching statistical significance. this observation again argues against the use of beta-actin as a reference when measuring gene expression in ccrcc, and requires one to reconsider ybx overexpression in most cases of our previous study. intriguingly, some reports suggest a possible role of actin expression in the tumorigenesis process. actin is a major component of cytoskeleton, and altered cell morphology is a characteristic of tumor cells, notably to enable invasiveness, migration and metastasis. for example, accumulation of beta-actin in tips of pseudopodia drives invasiveness and metastatic ability of transformed mdck cells. indeed, even though mechanisms are not yet well understood, changes in expression level of beta-actin were associated with higher invasiveness of hepatoma cells, and with metastatic potential of colon adenocarcinoma cell lines. besides its direct role in the control of cell morphology, actin dynamics could also regulate genetic programs by the so-called mechanogenetic mechanisms. indeed, ablation of beta-actin altered the ratio of globular actin to filamentous actin in mouse embryonic fibroblasts, with corresponding changes in expression of genes regulating cell cycle and motility. interestingly, except for one obvious outlier, variations of beta-actin expression in the cohort we analyzed appeared to correlate with ybx expression. variations of beta-actin expression also followed in parallel ybx expression during mouse kidney embryonic development. based on the well-known roles of ybx in the control of epithelium morphogenesis, one can hypothesize that ybx could regulate beta-actin expression during kidney ontogenesis and oncogenesis. expression of ybx3/zonab/csda as a function of tumor grade although we concluded above that ybx did not show systematic overexpression in the global cohort of ccrcc samples, a closer look suggested the possibility of three expression groups: high, intermediate, and low. indeed, as compared to matched normal tissues, five tumors exhibited a significant overexpression of ybx3; five other exhibited significant underexpression; while the six remaining did not show significant difference from their normal counterparts. we thus wondered if these variations in ybx levels could be related to ccrcc pathogenesis and more specifically to tumor grades and asked two experimented pathologists to independently grade the tumor samples according to fuhrman, being totally unaware of our expression level data. both pathologists agreed on the cases; one case was nevertheless excluded from further analysis as it contained 40% of normal parenchyma. of the remaining cases tumor samples were graded # were graded # and were graded # segmentation of ybx expression levels according to histopathological grading is shown in figure  this analysis revealed that ybx negatively correlates to tumor grade with higher expression levels in grade # intermediate in grade # and lower in grade #. this correlation was further analyzed at the protein level by immunohistology in tumor samples from grade # to #. ybx staining was clearly evident in all nuclei of grade # tumors. in grade # tumors, ybx staining was weaker in the nuclei and sometimes appeared in the cytoplasm. in grade # tumors, we observed strong inter- and intra-tumor heterogeneity of ybx staining; varying from complete absence to intense staining in some nuclei. these qualitative histopathological observations are in agreement with our rt-qpcr analyses showing decreased expression level of ybx from grade # to grade # and. even if these numbers were small, the negative correlation of ybx mrna expression level with tumor anaplasia, hence potential progression, was clearly significant. upregulation of ybx in grade # tumors, followed by progressive silencing in more anaplastic, hence more prone to metastasize, could indicate that ybx plays a biological role at a low degree of neoplastic transformation characterizing tumors with low metastatic potential, and be dispensable for disease progression. a similar trend has been recently reported in hepatocellular carcinoma. although ybx overexpression and nuclear localization was initially correlated to poor patients outcome, a recent study by the same team revealed that high ybx level in pre-cancerous “hypercarcinogenic” states, but not in established hcc, was, in fact, associated to poor prognosis. it is thus conceivable that high ybx expression would promote dedifferentiation and proliferation, thus secondarily favor mutations of cancer as a multistep process. an interesting alternative hypothesis is based on the ability of ybx to reduce tumor angiogenesis and lymphangiogenesis, so as to inhibit tumor growth and metastasis in lung cancer and squamous cell carcinoma models. the authors went so far as to propose ybx as a potential “therapeutic” gene, which, if artificially induced in tumors, could prevent disease progression and dissemination. of note, ybx is also down-regulated in breast cancer, as compared to healthy tissue. however, the grade of the breast cancer was not mentioned. if this interpretation is correct, ybx extinction in most anaplastic, hence potentially invasive tumor cells, would promote angio/lympho-genesis, thus favor metastatic dissemination.  in conclusion, although the pathogenic implications of ybx in human tumorigenesis clearly need to be further explored, its global mrna expression level may be part of a “signature” of rcc grading. competing interests the authors declare that no conflict of interest exists. authors’ contributions sd and asd conceived, designed, performed the experiments, analyzed results and drafted the manuscript. em and j-pc graded tumor samples and helped to draft the manuscript. pjc analyzed results and drafted the manuscript. cep supervised the study, analyzed the results and drafted the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 ybx correlation with known target genes and beta-actin. dot plots with respective mean fold variations of ybx3-Ψ and cycld, pcna, megalin/lrp- and b-act in the tumor samples compared to adjacent healthy tissues. red dotted lines indicate values in adjacent healthy tissues. black lines show linear regression curves. please note the logarithmic scale for megalin expression. spearman coefficient of correlation and p value are indicated. note that value of sample can be considered as outlier and was excluded. click here for file additional file 2 effects of primer and choice of reference hkgs on the estimation of normalized ybx expression in ccrcc samples. expression of ybx mrna, measured with ybx3 + Ψ and ybx3-Ψ primers, in each of the tumors compared to adjacent healthy tissue after normalization to either beta-actin, or ppia & rps. values are means of independent assays with standard deviations. tumor values statistically different from their normal paired tissues are indicated by asterisks. pairs to were those analyzed in our previous report. click here for file additional file 3 distribution of ybx3 + Ψ expression ratios in tumor samples according to histological grading. circles are values for ybx3 + Ψ expression ratios in the fuhrman-graded tumors compared to paired adjacent healthy tissue after normalization to geometric mean of ppia + rps lines are medians for each group and statistical differences between subgroups are indicated as well. the expression of ybx inversely correlates with tumor grade. click here for file acknowledgements the authors want to thank the biolibrary at the cancer centre of the cliniques universitaires saint-luc for their help in providing tissue samples and associated patients information. the work was supported by grants from the université catholique de louvain and the fund for scientific medical research. a.-s.d. is a doctoral fellow from télévie, sd is a post-doctoral fellow, and cep a research associate of the f.r.s.-fnrs.
we thus decided to re-evaluate ybx expression by real-time-pcr in a larger panel of ccrcc samples, and their paired healthy tissue, with special attention on experimental biases such as inter-individual variations, primer specificity, and reference gene for normalization. among tested housekeeping genes, ppia and rps especially in combination, proved best suitable to normalize gene expression in ccrcc tissues as compared to classical reference genes such as beta-actin, gapdh, 18s or b2m. this paper underlines that special attention to reference gene products in the design of real-time pcr analysis of tumoral tissue is crucial to avoid misleading conclusions. our laboratory reported overexpression of ybx in clear cell renal cell arcinoma, as part of a wide study of ybx regulation in vitro and in vivo. gene expression was measured by rt-qpcr in ccrcc samples, each compared to corresponding healthy tissue to minimize inter-individual variations. eight potential housekeeping genes were evaluated for expression level and stability among the 16-paired samples. using this pair as reference, ybx expression level among a collection of ccrcc tumors was not significantly increased as compared to normal adjacent tissues. however, stratification according to fuhrman grade disclosed higher ybx expression levels in low-grade tumors and lower in high-grade tumors. furthermore, we found that global ybx3/zonab/csda mrna expression level may be considered within a “signature” of rcc grading. immunoperoxidase confirmed homogeneous nuclear staining for ybx in low-grade but revealed nuclear heterogeneity in high-grade tumors. the preliminary data was limited to cases, of which only could be compared to paired normal tissue, and beta-actin was used as sole reference to normalize gene expression.
 ybx3/zonab/csda is an epithelial-specific transcription factor acting in the density-based switch between proliferation and differentiation. our laboratory reported overexpression of ybx in clear cell renal cell arcinoma, as part of a wide study of ybx regulation in vitro and in vivo. the preliminary data was limited to cases, of which only could be compared to paired normal tissue, and beta-actin was used as sole reference to normalize gene expression. we thus decided to re-evaluate ybx expression by real-time-pcr in a larger panel of ccrcc samples, and their paired healthy tissue, with special attention on experimental biases such as inter-individual variations, primer specificity, and reference gene for normalization.  gene expression was measured by rt-qpcr in ccrcc samples, each compared to corresponding healthy tissue to minimize inter-individual variations. eight potential housekeeping genes were evaluated for expression level and stability among the 16-paired samples. among tested housekeeping genes, ppia and rps especially in combination, proved best suitable to normalize gene expression in ccrcc tissues as compared to classical reference genes such as beta-actin, gapdh, 18s or b2m. using this pair as reference, ybx expression level among a collection of ccrcc tumors was not significantly increased as compared to normal adjacent tissues. however, stratification according to fuhrman grade disclosed higher ybx expression levels in low-grade tumors and lower in high-grade tumors. immunoperoxidase confirmed homogeneous nuclear staining for ybx in low-grade but revealed nuclear heterogeneity in high-grade tumors.  this paper underlines that special attention to reference gene products in the design of real-time pcr analysis of tumoral tissue is crucial to avoid misleading conclusions. furthermore, we found that global ybx3/zonab/csda mrna expression level may be considered within a “signature” of rcc grading. 