Paired Healthy Tissues Research Articles

Validation of housekeeping gene and impact on normalized gene expression in clear cell renal cell carcinoma: critical reassessment of YBX3/ZONAB/CSDA expression.

BackgroundYBX3/ZONAB/CSDA is an epithelial-specific transcription factor acting in the density-based switch between proliferation and differentiation. Our laboratory reported overexpression of YBX3 in clear cell renal cell arcinoma (ccRCC), as part of a wide study of YBX3 regulation in vitro and in vivo. The preliminary data was limited to 5 cases, of which only 3 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 YBX3 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.ResultsGene expression was measured by RT-qPCR in 16 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 RPS13, 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, YBX3 expression level among a collection of 16 ccRCC tumors was not significantly increased as compared to normal adjacent tissues. However, stratification according to Fuhrman grade disclosed higher YBX3 expression levels in low-grade tumors and lower in high-grade tumors. Immunoperoxidase confirmed homogeneous nuclear staining for YBX3 in low-grade but revealed nuclear heterogeneity in high-grade tumors.ConclusionsThis 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.

The Homeobox Gene MEIS1 Is Methylated in BRAFp.V600E Mutated Colon Tumors

Development of colorectal cancer (CRC) can occur both via gene mutations in tumor suppressor genes and oncogenes, as well as via epigenetic changes, including DNA methylation. Site-specific methylation in CRC regulates expression of tumor-associated genes. Right-sided colon tumors more frequently have BRAF p.V600E mutations and have higher methylation grades when compared to left-sided malignancies. The aim of this study was to identify DNA methylation changes associated with BRAF p.V600E mutation status. We performed methylation profiling of colon tumor DNA, isolated from frozen sections enriched for epithelial cells by macro-dissection, and from paired healthy tissue. Single gene analyses comparing BRAF p.V600E with BRAF wild type revealed MEIS1 as the most significant differentially methylated gene (log2 fold change: 0.89, false discovery rate-adjusted P-value 2.8*10-9). This finding was validated by methylation-specific PCR that was concordant with the microarray data. Additionally, validation in an independent cohort (n=228) showed a significant association between BRAF p.V600E and MEIS1 methylation (OR: 13.0, 95% CI: 5.2 - 33.0, P<0.0001). MEIS1 methylation was associated with decreased MEIS1 gene expression in both patient samples and CRC cell lines. The same was true for gene expression of a truncated form of MEIS1, MEIS1 D27, which misses exon 8 and has a proposed tumor suppression function. To trace the origin of MEIS1 promoter methylation, 14 colorectal tumors were flow-sorted. Four out of eight BRAF p.V600E tumor epithelial fractions (50%) showed MEIS1 promoter methylation, as well as three out of eight BRAF p.V600E stromal fractions (38%). Only one out of six BRAF wild type showed MEIS1 promoter methylation in both the epithelial tumor and stromal fractions (17%). In conclusion, BRAF p.V600E colon tumors showed significant MEIS1 promoter methylation, which was associated with decreased MEIS1 gene expression.

Proteomic analysis of formalin-fixed paraffin-embedded colorectal cancer tissue using tandem mass tag protein labeling

In clinical research, repositories of biological samples form a rich source of clinical material for biomarker studies. Banked material, however, is often not stored in optimal conditions regarding the technology used for biomarker research. A case in point is formalin-fixed paraffin-embedded (FFPE) tissue that could be used to obtain large cohorts of samples over a short period of time, as these tissues are routinely prepared for pathological analysis. However, in the context of mass spectrometry based peptide-centric proteomics, protein extraction and identification can be hampered by formalin-induced crosslinking. Furthermore, the molecular formalin crosslinks might be entangled differently across various samples, making it more difficult to reproducibly extract the same proteins from different samples. In this study, we establish the crosslink variability using Tandem Mass Tag (TMT) protein labeling followed by digestion, separation, identification and quantification of proteins extracted from FFPE colorectal cancer and paired healthy tissues. Moreover, by applying de novo interpretation of tandem mass spectra and subsequent analysis by Peaks PTM, unspecified modifications could be elucidated, leading to increased protein and proteome coverage. This approach might be useful for future FFPE proteomics studies.

Zymographic analysis of circulating and tissue forms of colon carcinoma gelatinase A (MMP-2) and B (MMP-9) separated by mono- and two-dimensional electrophoresis.

Gelatinase A (MMP-2) and gelatinase B (MMP-9) play a key role in the proteolytic cascade leading to ECM degradation during invasion and metastasis. The enzyme activity is regulated both at the intra- and extra-cellular level. Extracellular regulation is achieved mainly through the balance between proenzyme activation and inhibition, which appears to be altered in cancer patients. One of the mechanisms of MMP inhibition is the binding of the enzymes to appropriate tissue inhibitors (TIMP). In the recent literature, it has been suggested that MMP-2 and/or MMP-9 are indeed over-produced in many carcinomas, while the identity of the various enzymatic forms (latent, activated and enzyme/inhibitor complexes) remains to be elucidated. In this study we have analyzed the circulating forms of MMP-9 and MMP-2 in serum samples of patients with colon carcinoma, as well as the enzymatic activities present in tissue extracts from surgical fragments (primary tumor and its paired healthy tissue). Proteins were separated by means of mono-dimensional or bidimensional electrophoresis, and the enzymes detected by gelatin zymography and immunological assays. The results of densitometric analyses demonstrate that proMMP-9, but not proMMP-2, is significantly higher in the oncologic sera vs. the normal sera. In addition, several oligomeric circulating and tissue forms of MMP-9 are preferentially found in the oncologic samples, both in mono- and second-dimension zymograms. The activated forms of MMP-2 and MMP-9 are uniquely present in the primary tumor extracts, thus confirming the involvement of the tissue microenvironment in gelatinase activation and function.