Spotlight on Differentially Expressed Genes in Urinary Bladder Cancer

INTRODUCTION: We previously identified common differentially expressed (DE) genes in bladder cancer (BC). In the present study we analyzed in depth, the expression of several groups of these DE genes. MATERIALS AND METHODS: Samples from 30 human BCs and their adjacent normal tissues were analyzed by whole genome cDNA microarrays, qRT-PCR and Western blotting. Our attention was focused on cell-cycle control and DNA damage repair genes, genes related to apoptosis, signal transduction, angiogenesis, as well as cellular proliferation, invasion and metastasis. Four publicly available GEO Datasets were further analyzed, and the expression data of the genes of interest (GOIs) were compared to those of the present study. The relationship among the GOI was also investigated. GO and KEGG molecular pathway analysis was performed to identify possible enrichment of genes with specific biological themes. RESULTS: Unsupervised cluster analysis of DNA microarray data revealed a clear distinction in BC vs. control samples and low vs. high grade tumors. Genes with at least 2-fold differential expression in BC vs. controls, as well as in non-muscle invasive vs. muscle invasive tumors and in low vs. high grade tumors, were identified and ranked. Specific attention was paid to the changes in osteopontin (OPN, SPP1) expression, due to its multiple biological functions. Similarly, genes exhibiting equal or low expression in BC vs. the controls were scored. Significant pair-wise correlations in gene expression were scored. GO analysis revealed the multi-facet character of the GOIs, since they participate in a variety of mechanisms, including cell proliferation, cell death, metabolism, cell shape, and cytoskeletal re-organization. KEGG analysis revealed that the most significant pathway was that of Bladder Cancer (p = 1.5×10(-31)). CONCLUSIONS: The present work adds to the current knowledge on molecular signature identification of BC. Such works should progress in order to gain more insight into disease molecular mechanisms

Spotlight on Differentially Expressed Genes in Urinary Bladder Cancer

Introduction: We previously identified common differentially expressed (DE) genes in bladder cancer (BC). In the present study we analyzed in depth, the expression of several groups of these DE genes. Materials and Methods: Samples from 30 human BCs and their adjacent normal tissues were analyzed by whole genome cDNA microarrays, qRT-PCR and Western blotting. Our attention was focused on cell-cycle control and DNA damage repair genes, genes related to apoptosis, signal transduction, angiogenesis, as well as cellular proliferation, invasion and metastasis. Four publicly available GEO Datasets were further analyzed, and the expression data of the genes of interest (GOIs) were compared to those of the present study. The relationship among the GOI was also investigated. GO and KEGG molecular pathway analysis was performed to identify possible enrichment of genes with specific biological themes. Results: Unsupervised cluster analysis of DNA microarray data revealed a clear distinction in BC vs. control samples and low vs. high grade tumors. Genes with at least 2-fold differential expression in BC vs. controls, as well as in non-muscle invasive vs. muscle invasive tumors and in low vs. high grade tumors, were identified and ranked. Specific attention was paid to the changes in osteopontin (OPN, SPP1) expression, due to its multiple biological functions. Similarly, genes exhibiting equal or low expression in BC vs. the controls were scored. Significant pair-wise correlations in gene expression were scored. GO analysis revealed the multi-facet character of the GOIs, since they participate in a variety of mechanisms, including cell proliferation, cell death, metabolism, cell shape, and cytoskeletal re-organization. KEGG analysis revealed that the most significant pathway was that of Bladder Cancer (p = 1.5x10(-31)). Conclusions: The present work adds to the current knowledge on molecular signature identification of BC. Such works should progress in order to gain more insight into disease molecular mechanisms

Transcription Factor Binding Motifs, Chromosome mapping and Gene Ontology analysis in Cross-platform microarray data from bladder cancer.

<p>We have previously analyzed the gene expression profile in urinary bladder cancer and determined the differentially expressed (DE) genes between cancer and healthy tissue. We aimed: 1) To identify the over-represented Transcription Factor Binding Motifs (TFBMs) in the promoters of the DE genes. 2) To map the DE genes on the chromosomal regions. 3) To gain more insight into the DE gene functions, using Gene Ontology (GO) analysis. We investigated the TFBMs in the Transcription Element Listening System Database (TELiS). The TRANSFAC TF database was used for the identification of TF binding sites. The Gene Ontology Tree Machine, WebGestalt web-tool and the Matlab ® (The Mathworks Inc.) computing environments were used for chromosome mapping. GO analysis was performed using the eGOn online tool. The WebGestalt web-tool was used for gene function classifications. Relations of the DE genes and the transcription factor binding motifs were further investigated using the Pubgene Ontology Database. The glucocorticoid receptor (GR) was predicted as one of the TFs in the common gene set. In order to find which gene was most commonly represented among the TFs, we plotted the incidence of each gene as a function of the times of appearance within the predicted TFs. The gene BMP4 (bone morphogenetic protein 4; ID: 652) exhibited the higher number of binding sites for the predicted TFs. The majority of the chromosomes in BC had inactivated (down-regulated) genes, compared to the normal tissue. However, two genes were significantly over-expressed: CDC20 (in chromosome 1) and HCCS (in chromosome X). Three main functions were outlined by GO for the DE genes: a) circulatory system regulation, b) reproductive organ and sex development, and c) catecholamine metabolism. This enrichment showed that the predicted gene set has more than a dual role. Through this study, we were able to identify several important factors that warrant further investigation both as prognostic markers and as therapeutic targets for bladder cancer. Such approaches may provide a better insight into tumorigenesis and tumor progression.</p

Linear correlations in chromosomal-based gene expression in urinary bladder cancer

<p>Introduction & Objectives: Gene expression is a very tidy and well coordinated procedure. Consecutive genes are often similarly expressed. We hypothesized that correlations might exist between genes of the same chromosome, yet belonging to different urinary bladder cancer (BC) samples, in order to indicate a common regulation for genes following this pattern.</p> <p>Materials & Methods: We analyzed BC gene expression profiles, with emphasis in linear correlations of gene expression based on their chromosomal locations. Samples from 10 human BCs and 5 normal tissues were analyzed by whole genome microarrays, along with a computational approach, for their expression profiles. After raw data normalization and classification, differentially expressed genes (DE) were sorted according to their chromosome distributions and were further investigated for linear correlations among them. Chromosomal activity in terms of gene expression was measured by calculating the average expression of all DE genes for each chromosome, both for tumour and control samples.</p> <p>Results: Chromosome-based expression analysis predicted that among the most active chromosomes were chromosomes 9 and X. Similarly, control samples also manifested high expression activity on the X chromosome. The genes that exhibited significant linear correlations (p<0.05) among tumor samples on chromosomes 4, 8, 13, 21 and 22, were as follows: TACR3, RNF150, ANXA10, CENTD1, EXOC1, GRSF1 for chromosome 4; ANXA13, DENND3, FGF20, EFHA2, DNAJC5B, MRPS28, FABP5 for chromosome 8; ITGBL1, RXFP2, KL, MYCBP2, FARP1 for chromosome 13; KRTAP19-1, IFNAR1, SON for chromosome 21; MORC2, PLA2G6, ACO2, ARHGAP8 for chromosome 22; SERPINA7, TMEM164, ARHGAP6, APLN, FHL1, PNMA6A, UBL4A, PRDX4, POLA1, MXRA5 for chromosome X.</p> <p>Conclusions: Despite the fact that linear correlations occurred among distinct patients, the expression of the genes appeared to be correlated among them, in a similar manner. We have previously reported that there are hints of common mechanisms between BCs of different stage/grade, employing microarray analysis. Chromosomal correlation analysis comes to support our previous findings, since it revealed genes bearing common regulation among samples of different histology. Gene expression correlations can further assist us to understand more in-depth the mechanisms underlying tumour progression and biology.</p

Cross-platform comparisons of microarray data. Elucidation of common differentially expressed genes in bladder cancer.

<p>INTRODUCTION: Parallel gene-expression monitoring is a powerful tool for analyzing relationships among tumors, discovering new tumor subgroups, assigning tumors to pre-defined classes, identifying co-regulated or tumor stage-specific genes and predicting disease outcome. Previous gene expression studies have focused on identifying differences between tumor samples of the same type.</p> <p>AIM OF STUDY: Using a reverse engineering approach, we searched for common expression profiles among tumor samples. We analyzed the gene expression profile of bladder cancer (BC) and determined the differentially expressed (DE) genes between cancer and healthy tissue, using cross-platform comparisons.</p> <p>MATERIALS AND METHODS: We performed cDNA microarray analysis, comprising both in-house experimental and publicly available GEO microarray data. In total, our pooled microarray analysis was composed of 17 control samples (n=5, for the CodeLink platform; and n=12, for the remaining microarray platforms) and 129 BC samples (n=10, for the CodeLink platform; and n=119, for the remaining microarray platforms). Tumor samples were separated into the following groups: Ta/T1 without CIS; Ta/T1 with CIS; Ta-grade 1; Ta-grade 3; T1-grade2; T1-grade 3; T2-grade 2-4. Each group was compared against all control samples and the DE genes were identified. Data were clustered with different algorithms.</p> <p>RESULTS:</p> <p>A two-sample T-test analysis for all tumor samples vs. all normal samples, revealed 434 DE genes between the two tissue groups. Hierarchical clustering (HCL) showed a clear distinction among tumor samples. In total, 17 genes appeared to be commonly expressed among all BC samples: BMP4, CRYGD, DBH, GJB1, KRT83, MPZ, NHLH1, TACR3, ACTC1, MFAP4, SPARCL1, TAGLN, TPM2, CDC20, LHCGR, TM9SF1 and HCCS. Three groups of genes were down-regulated in all samples: BMP4, CRYGD, DBH, GJB1, KRT83, MPZ, NHLH1, TACR3 in cluster 79; ACTC1, MFAP4, SPARCL1, TAGLN in cluster 81; and TPM2 in cluster 82. CDC20, TM9SF1 and HCCS appeared to be simultaneously over-expressed in all tumor groups. LHCGR was differentially expressed in 108/129 (83.7%) of the BC samples.</p> <p>DISCUSSION: Through this investigation we were able to identify several important factors that warrant further investigation both as prognostic markers and as therapeutic targets. Such approaches may provide a better insight into tumorigenesis and tumor progression.</p

MiR-21 can be used as independent prognostic factor for survival and metastasis in urinary bladder cancer.

<p>Introduction & Objectives: Our goal was to correlate the expression of 12 micro-RNAs with the corresponding expression of FGF2, OPN and VEGFA. Gene expression was correlated with the overall and cancer-specific survival of patients suffering from urinary bladder cancer (BC), as well as with recurrence and metastasis.</p> <p>Materials & Methods: Gene expression were acquired by qPCR, from 77 BC specimens. Correlation of the gene expression with survival, recurrence and metastasis was employed by SPSS.</p> <p>Results: High expression of miR-21 correlated with worse overall survival (p=0.0099). Univariate analysis showed that miR-21 and miR-210 can be used as independent prognostic factors for overall survival (p=0.015 and p=0.049, respectively). Moreover, univariate analysis revealed that miR-21 can be used as independent prognostic factor for metastasis (p=0.049). Multivariate analysis revealed that miR-21, miR-210 and miR-378_1 can be used as independent prognostic factors for overall survival (p=0.005, p=0.033 and p=0.012, respectively); miR-21 and miR-378_1 can be used as independent prognostic factors for recurrence (p=0.030 and p=0.031, respectively); and miR-21 can be used as independent prognostic factors for metastasis (p=0.049). FGF2 was positively correlated with the majority of the miRs both in BC and normal tissue (p<0.001). OPN was positively correlated with miR-145_1 (p=0.015) in BC, and with miR-296-5p (p=0.017) in normal tissue. VEGFA was positively correlated with miR-21 in BC (p=0.043), and with miR-205_1 (p=0.045), FGF2 (p=0.004) and OPN (p<0.001) in normal tissue.</p> <p>Conclusions: miR-21 can be used as independent prognostic factor both for overall patient survival and metastasis of BC. miR-210 is an independent prognostic factor for overall survival.</p

microRNA implication in urinary bladder cancer.

<p>INTRODUCTION: microRNAs (miRNAs) are a class of ~22 nt non-coding RNAs that regulate gene expression post-transcriptionally. The oncogenic miRNAs (oncomiRs) display anti-apoptotic activity and are over-expressed in cancer cells, whereas, miRNAs with anti-proliferative and pro-apoptotic activity function as tumor-suppressor genes and are under-expressed in cancer cells.</p> <p>AIM OF STUDY: Our goal was to investigate the expression profile of a various oncomiRs and tumor-suppressor miRs in urinary bladder cancer (BC).</p> <p>MATERIALS AND METHODS: Seventy-seven urinary BC cases, along with 77 matched tumor-associated normal samples were investigated for the expression of 11 miRNAs using qPCR. The relationship among the expression of miR-10b, miR-19a, miR-19b, miR-21, miR-126, miR-145, miR-205, miR-210, miR-221, miR-296-5p and miR-378-1, as well as with the pathologic features of the tumors, was further examined. The influence of miR expression on the overall and cancer-specific survival of the patients, as well as putative target genes of the up- and down-regulated miRNAs were also predicted. RESULTS: As expected, the majority of the microRNAs studied (miR-10b, miR-19a, miR-19b, miR-126_1, miR-145, miR-221, miR-296-5p and miR-378-1) exhibited significant down-regulation in BC vs. the normal tissue. A great range in the x-fold expression values of all microRNAs was noticed. High expression of miR-21 correlated with worse overall survival (p=0.0099). Univariate analysis showed that miR-21 and miR-210 can be used as independent prognostic factors for overall survival (p=0.015 and p=0.049, respectively). Moreover, univariate analysis revealed that miR-21 can be used as independent prognostic factor for metastasis (p=0.049). Multivariate analysis revealed that miR-21, miR-210 and miR-378 can be used as independent prognostic factors for overall survival (p=0.005, p=0.033 and p=0.012, respectively); miR-21 and miR-378 can be used as independent prognostic factors for recurrence (p=0.030 and p=0.031, respectively); and miR-21 can be used as independent prognostic factors for metastasis (p=0.049).</p> <p>DISCUSSION: The data reported here demonstrate that several microRNAs are deregulated in BC through down-regulation. miR-21 can be used as independent prognostic factor both for patient survival and metastasis. miR-210 can be used as an independent prognostic factor for overall survival.</p

Expression profile of oncomiRs and tumor-suppressor miRs in urothelial carcinoma of the bladder.

<p>Introduction & Objectives: Micro-RNAs are small, regulatory molecules approximately 21-24 nucleotides in length. They function at the post-transcriptional level by controlling the expression of more than 50% of human protein-coding genes and play an essential role in cell signaling pathways. Our goal was explore the expression profile of oncomiRs and tumor-suppressor miRs, and to define their possible correlations in urothelial carcinoma of the bladder (BC).</p> <p>Materials & Methods: Seventy-seven primary BCs, along with 77 matched tumor-associated normal samples were investigated for the expression of 12 micro-RNAs using qPCR. Relationships between the expression of miR-10b, miR19a, miR19b, miR-21, miR-122a, miR-145_1, miR-205_1, miR-210, miR-221, miR-222, miR-378-1 and miR-296-5p and the pathologic features of the tumors were also examined.</p> <p>Results: The majority of the micro-RNAs exhibited down-regulation in BC vs. normal tissue [miR-10b (p=0.0007), miR-19a (p=0.012), miR-19b (p=0.0361), miR-126_1 (p=0.0021), miR-145_1 (p<0.0001), miR-221 (p<0.0001), miR-296-5p (p<0.0001), miR-378-1 (p<0.0001)]. miR-21, miR-205_1 and miR-210 expression levels did not present difference between BC and normal tissue. However, we noticed a great range in the x-fold expression values of all micro-RNAs. The median x-fold expression (range) was as follows: miR-10b, 0.45 (0-12.58); miR-19a, 0.56 (0-25.63); miR-19b, 0.50 (0-18.90); miR-21, 0.96 (0-52.95); miR-126_1, 0.36 (0-42.62); miR-145_1, 0.04 (0-56.36); miR-205_1, 1.07 (0.01-36.42); miR-210, 1.09 (0-44.43); miR-221, 0.32 (0-33.51); miR-296-5p, 0.08 (0-75.24); miR-378-1, 0.17 (0-3.66). Significant correlations among all of the studied microRNAs were scored both in BC and control tissue.</p> <p>Conclusions: Different micro-RNAs are deregulated in BC through down-regulation. A synergistic involvement of these genes in the development of BC is implied.</p