Complementary Deoxyribonucleic Acid Microarray Research Articles

Shared Genes and Molecular Mechanisms between Nonalcoholic Fatty Liver Disease and Hepatocellular Carcinoma Established by WGCNA Analysis.

Background Hepatocellular carcinoma (HCC) is one of the leading causes of death from cancer worldwide. The histopathological features, risk factors, and prognosis of HCC caused by nonalcoholic fatty liver disease (NAFLD) appear to be significantly different from those of HCC caused by other etiologies of liver disease. Objective This article explores the shared gene and molecular mechanism between NAFLD and HCC through bioinformatics technologies such as weighted gene co-expression network analysis (WGCNA), so as to provide a reference for comprehensive understanding and treatment of HCC caused by NAFLD. Methods NAFLD complementary deoxyribonucleic acid microarrays (GSE185051) from the Gene Expression Omnibus database and HCC ribonucleic acid (RNA)-sequencing data (RNA-seq data) from The Cancer Genome Atlas database were used to analyze the differentially expressed genes (DEGs) between NAFLD and HCC. Then, the clinical traits and DEGs in the two disease data sets were analyzed by WGCNA to obtain W-DEGs, and cross-W-DEGs were obtained by their intersection. We performed subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) enrichment analyses of the cross-W-DEGs and established protein-protein interaction networks. Then, we identified the hub genes in them by Cytoscape and screened out the final candidate genes. Finally, we validated candidate genes by gene expression, survival, and immunohistochemical analyses. Results The GO analysis of 79 cross-W-DEGs showed they were related mainly to RNA polymerase II (RNAP II) and its upstream transcription factors. KEGG analysis revealed that they were enriched predominantly in inflammation-related pathways (tumor necrosis factor and interleukin-17). Four candidate genes (JUNB, DUSP1, NR4A1, and FOSB) were finally screened out from the cross-W-DEGs. Conclusion JUNB, DUSP1, NR4A1, and FOSB inhibit NAFLD and HCC development and progression. Thus, they can serve as potential useful biomarkers for predicting and treating NAFLD progression to HCC.

Collaborative Priors with SVD for Denoising of cDNA Microarray Images

Background/Objectives: This study presents an external and internal prior guided patch based filter for minimizing Gaussian noise in complementary deoxyribonucleic acid (cDNA) microarray images. Methods/Statistical analysis: The proposed denoising filter is capable of taking into consideration both external and internal prior. It employs combined prior guided patch based denoising considering various distance based patch-matching methods. Findings: Experimental results demonstrate that the combined prior guided patch based filter outperforms the existing well-known filters in minimizing noise of cDNA microarray images. The outcome of the proposed scheme found to offer better peak signalto-noise ratio and structural similarity index in contrast to existing filtering techniques. Effectiveness of the proposed denoising method is also assessed by estimating the spot intensities of cDNA microarray image that reflects the effect of noise reduction in the image. Application/Improvements: Minimization of noise is a crucial step of cDNA microarray image processing and it aids in microarray analysis by extracting valid and good quality gene expression measurements. Keywords: Denoising, External Prior, Internal Prior, K- nearest Neighbor, Microarray Image, Singular Value Decomposition

A COMPARATIVE STUDY OF NOISE REMOVAL IN CDNA MICROARRAY IMAGE USING DIFFERENT FILTERING TECHNIQUES

In complementary Deoxyribonucleic Acid (cDNA) Microarray image noises usually occur during the accusation or while transmission. It is necessary to remove the noise to provide further processing techniques like edge detection, segmentation, etc. In this paper we gives comparative analysis to remove salt and pepper noise and speckle noise using different filtering techniques like standard median filter, switched median filter and vector median filter. The Mean Squared Error (MSE) and Peak Signal to Noise Ratio (PSNR) values are calculated for different density of salt and pepper noise and speckle noise. To find the better noise removal technique the comparison is done based on high PSNR value for different filtering techniques. The MATLAB based simulation is done to calculating the MSE and PSNR values.

Acceleration of healing of the medial collateral ligament of the knee by local administration of synthetic microRNA-210 in a rat model

BackgroundInjury to the medial collateral ligament (MCL) of the knee joint is the most common ligament injury of the knee. Ligament healing generally takes a long time. Micro-ribonucleic acid (miRNA) is one of the noncoding RNAs and plays a crucial role in physiological function; miRNA (miR)-210 is known as a potent factor of angiogenesis, which is an important initiator of ligament healing. The purpose of this study is to examine the effect of local injection of double-stranded (ds) miR-210 on the healing of the MCL of rat knee joint. MethodsMCLs of Sprague-Dawley rats were cut transversely. After the fascia and skin were sutured, dsmiR-210 or control dsRNA was injected into the injured site of MCL. At 2 weeks and 4 weeks, histological analysis and immunofluorescence staining of vascular endothelial growth factor, isolectin B4, collagen type 1, and Ki67 as well as a mechanical test were performed. Analysis of complementary deoxyribonucleic acid (cDNA) microarray data was performed at 1 week. ResultsHistological analysis showed that parallel fibres in the injured site were organised at 2 weeks and became thicker at 4 weeks in the miR-210-treated group, whereas the injured site in controls was filled with loose fibrous tissues and was thinner than that in the miR-210-treated group. The number of blood vessels in the miR-210-treated group was significantly higher than that in controls (p < 0.05), and vascular endothelial growth factor, Ki67, and collagen type 1 in the miR-210-treated group were intensely expressed in the repaired site as compared to the control group. The mechanical test indicated that the ultimate failure load in the miR-210-treated group was significantly higher than that in the control group at 2 weeks. The cDNA microarray analysis showed significant upregulation of genes related to cell proliferation and cell differentiation, and genes involved in negative regulation of apoptosis. ConclusionThis study showed that local injection of dsmiR-210 could accelerate MCL healing in rat, which is likely due to stimulation of angiogenesis at the healing site.

Steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation

OBJECTIVE:To analyze steroidogenesis-related gene expression in the rat ovary exposed to melatonin supplementation.METHODS:Thirty-two virgin adult female rats were randomized to two groups as follows: the control group GI received vehicle and the experimental group GII received melatonin supplementation (10 µg/night per animal) for 60 consecutive days. After the treatment, animals were anesthetized and the collected ovaries were immediately placed in liquid nitrogen for complementary deoxyribonucleic acid microarray analyses. A GeneChip® Kit Rat Genome 230 2.0 Affymetrix Array was used for gene analysis and the experiment was repeated three times for each group. The results were normalized with the GeneChip® Operating Software program and confirmed through analysis with the secondary deoxyribonucleic acid-Chip Analyzer (dChip) software. The data were confirmed by real-time reverse transcription polymerase chain reaction analysis. Genes related to ovarian function were further confirmed by immunohistochemistry.RESULTS:We found the upregulation of the type 9 adenylate cyclase and inhibin beta B genes and the downregulation of the cyclic adenosine monophosphate response element modulator and cytochrome P450 family 17a1 genes in the ovarian tissue of GII compared to those of the control group.CONCLUSION:Our data suggest that melatonin supplementation decreases gene expression of cyclic adenosine monophosphate, which changes ovarian steroidogenesis.

Using fuzzy logic and particle swarm optimization to design a decision-based filter for cDNA microarray image restoration

This paper presents a Decision-Based cDNA Microarray Image filter for the removal of impulse noises for complementary deoxyribonucleic acid (cDNA) microarray images. It employs Fuzzy Logic (FL) technique to design a noise detector optimized by Particle Swarm Optimization (PSO), and utilizes center weighted vector median (CWVM) filter for restoring. For convenience, it is called the DBAMI filter hereafter. The proposed filter first employs the FL technique to detect each vector-valued pixel of a cDNA microarray image. If it is considered as noise-corrupted, the DBAMI filter restores it with the CWVM filter. Otherwise, it remains unchanged. Experimental results demonstrate that the DBAMI filter outperforms the existing well-known filters in restoration of cDNA microarray images under considerations here.

The Vav3 oncogene enhances the malignant potential of prostate cancer cells under chronic hypoxia

ObjectivesWe had previously reported that chronic hypoxia induces androgen-independent growth in the human prostate cancer cell line LNCaP. In this study, we have identified a key molecule, the Vav3 oncogene, and investigated the effects of Vav3 overexpression on cancer cell growth and malignant behavior and the possible apoptosis-inducing effect of Vav3 expression knockdown by small interfering ribonucleic acid (siRNA) in LNCaP cells under chronic hypoxia (LNCaP/CH). Methods and materialsHypoxia-inducible oncogenes were identified by complementary deoxyribonucleic acid (cDNA) microarray and Ingenuity Pathway Analysis in order to investigate gene ontology and functional pathways and networks. siRNA was used to knockdown the Vav3 target gene and analyze the effects on proliferation, invasion, migration, and apoptosis of LNCaP/CH cells. Vav3 cDNA was transfected into LNCaP cells under normoxia (LNCaP/N) to establish Vav3-overexpressing clonal cell lines, whose proliferation, invasion, and migration was then examined. Immunoblot analysis was used to investigate the activation of Akt, a Vav3 downstream target molecule. ResultscDNA microarray analysis and Ingenuity Pathway Analysis identified Vav3 as a hypoxia-inducible oncogene that was highly associated with malignant behavior. Vav3 messenger RNA and protein expression in LNCaP/CH cells were higher than in LNCaP/N and LNCaP cells cultured under acute hypoxia (LNCaP/AH). The growth rate of LNCaP/CH cells was lower than that of LNCaP/N cells but higher than that of LNCaP/AH cells. LNCaP/CH cells showed higher invasion and migration than LNCaP/N and LNCaP/AH cells. Interrupting Vav3 expression strongly suppressed the proliferation, invasion, and migration of LNCaP/CH cells. Furthermore, siRNA led to apoptosis with increased caspase-3 and cleaved poly (adenosine diphosphate-ribose) polymerase activation in LNCaP/CH cells. Stable Vav3 overexpression in LNCaP cells promoted cell proliferation, invasion, and migration with Akt activation. ConclusionsOur results demonstrate that Vav3 plays a crucial role in prostate cancer growth and malignant behavior, thus revealing a novel potential therapeutic target.

Probing Regulatory Proteins for Vascular Contraction by Deoxyribonucleic Acid Microarray

Background and ObjectivesThe heat-shock response modulates contractility of vascular smooth muscles. With complementary deoxyribonucleic acid microarray, we tried to identify the novel genes that are involved in the regulation of vascular contraction after heat shock.Materials and MethodsHuman radial artery strips were mounted in organ baths, exposed at 42℃ for 45 minutes, and returned to equilibrate at 37℃. This study examined gene expression profile associated with heat-shock response in radial arteries of patients with hyperlipidemia by using a microarray that contained 5763 human cDNA. The results of microarray hybridization experiments from the radial arteries of 4 different subjects were analyzed and classified by the cluster program.ResultsAmong these differentially-expressed genes, Hsp70, Hsp10, αB-crystallin, and Hsp60 were significantly increased by the heat shock response. Of non-HSP genes, 15 genes increased, while 22 genes decreased. Among these 37 genes, αB-crystallin (CRYAB) (up 1.92-fold), myosin, light polypeptide kinase transcript variant 8, 6 (up 1.70-fold, up 1.68-fold), catenin (cadherin-associated protein, alpha-like 1) (down-0.57 fold) and tropomyosin 3 (down 0.68-fold) were thought to be related with the contraction. Real-time quantitative polymerase chain reaction showed that Hsp70, Hsp10 and αB-crystallin were significantly increased.ConclusionGene expression profile by heat shock provides information about genes implicated in augmentation of vascular contraction after heat shock.

Genome‐wide transcriptome analysis of fluoroquinolone resistance in clinical isolates of Escherichia coli

Coincident with their worldwide use, resistance to fluoroquinolones in Escherichia coli has increased. To identify the gene expression profiles underlying fluoroquinolone resistance, we carried out genome-wide transcriptome analysis of fluoroquinolone-sensitive E. coli. Four fluoroquinolone-sensitive E. coli and five fluoroquinolone-resistant E. coli clinical isolates were subjected to complementary deoxyribonucleic acid microarray analysis. Some upregulated genes' expression was verified by real-time polymerase chain reaction using 104 E. coli clinical isolates, and minimum inhibitory concentration tests were carried out by using their transformants. A total of 40 genes were significantly upregulated in fluoroquinolone-resistant E. coli isolates (P < 0.05). The expression of phage shock protein operons, which are involved in biofilm formation, was markedly upregulated in our profile of fluoroquinolone-resistant E. coli. One of the phage shock protein operons, pspC, was significantly upregulated in 50 fluoroquinolone-resistant E. coli isolates (P < 0.0001). The expression of type I fimbriae genes, which are pilus operons involved in biofilm formation, were markedly downregulated in fluoroquinolone-resistant E. coli. Deoxyribonucleic acid adenine methyltransferase (dam), which represses type I fimbriae genes, was significantly upregulated in the clinical fluoroquinolone-resistant E. coli isolates (P = 0.007). We established pspC- and dam-expressing E. coli transformants from fluoroquinolone-sensitive E. coli, and the minimum inhibitory concentration tests showed that the transformants acquired fluoroquinolone resistance, suggesting that upregulation of these genes contributes to acquiring fluoroquinolone resistance. Upregulation of psp operones and dam underlying pilus operons downregulation might be associated with fluoroquinolone resistance in E. coli.

POU6F1 is the transcription factor that might be involved in cell proliferation of clear cell adenocarcinoma of the ovary

Clear cell adenocarcinoma of the ovary often shows resistance to anticancer agents. We investigated new molecules to use when developing molecular-targeting therapy for clear cell adenocarcinoma of the ovary. RMG-I cells without invasive potential and RMG-V cells with invasive potential (derived from clear cell adenocarcinoma of the ovary) were subjected to complementary deoxyribonucleic acid microarray analysis. Caveolin-1, a molecule involved in cellular motility and invasion, showed differing expression between the two cell lines. An RNA interference experiment using the published siRNA for caveolin-1 was carried out. The results showed suppression of RMG-V cell infiltration by siRNA, but proliferation of the cancer cells was also suppressed. In other words, RMG-V cell infiltration may have been suppressed simply because cell proliferation was suppressed by RNA interference. These findings suggested that POU6F1 might be a transcription factor involved in the proliferation of ovarian cancer cells. Clear cell adenocarcinoma of the ovary shows little response to standard therapy. The results of the present study suggest that the transcription factor POU6F1 could be a new molecular target for treatment of this cancer.

Integrated transcriptomic response to cardiac chronic hypoxia: translation regulators and response to stress in cell survival

Complementary deoxyribonucleic acid microarray data from 36 mice subjected for 1, 2, or 4 weeks of their early life to normal atmospheric conditions (normoxia) or chronic intermittent (CIH) or constant (CCH) hypoxia were analyzed to extract organizational principles of the developing heart transcriptome and determine the integrated response to oxygen deprivation. Although both CCH and CIH regulated numerous genes involved in a wide diversity of processes, the changes in maturational profile, expression stability, and coordination were vastly different between the two treatments, indicating the activation of distinct regulatory mechanisms of gene transcription. The analysis focused on the main regulators of translation and response to stress because of their role in the cardiac hypertrophy and cell survival in hypoxia. On average, the expression of each heart gene was tied to the expression of about 20% of other genes in normoxia but to only 8% in CCH and 9% in CIH, indicating a strong decoupling effect of hypoxia. In contrast to the general tendency, the interlinkages among components of the translational machinery and response to stress increased significantly in CIH and much more in CCH, suggesting a coordinated response to the hypoxic stress. Moreover, the transcriptomic networks were profoundly and differently remodeled by CCH and CIH.

Upregulation of the constitutively expressed HSC70 by KLF4

Krüppel-like factor 4 (KLF4) is a transcription factor that is abundantly expressed in various organisms from bacteria to mammals. It has been demonstrated that KLF4 regulates the expression of a wide range of genes. Analysis of KLF4 target genes reveals its diverse regulatory functions in cell growth, proliferation, differentiation, embryogenesis, and inflammation. However, the regulation of the expression of inducible heat shock protein 70 (HSP72) and heat shock cognate 70 (HSP73) by KLF4 is not defined. In our previous study, a complementary deoxyribonucleic acid microarray assay showed that KLF4 overexpression led to dramatic upregulation of HSP73 messenger ribonucleic acid (mRNA) in murine C2C12 myoblast cells, suggesting that HSP73 is a potential target gene regulated by KLF4. The effect of KLF4 on the expression of HSP72 and HSP73 was further examined by reverse transcriptase polymerase chain reaction and Western blot in KLF4-overexpressing or KLF4-deficient cells. The results showed the upregulation of the HSP73 constitutive expression by KLF4 overexpression in both C2C12 cells and murine RAW264.7 macrophages; in response to heat stress, however, few changes were observed in the levels of HSP73 by KLF4 overexpression. In addition, knockdown of endogenous KLF4 expression by morpholino antisense oligonucleotides significantly decreased both HSP73 mRNA and protein levels under normal conditions. Conversely, KLF4 had no effect on the expression of HSP72. Taken together, these findings suggest an important role for KLF4 as a novel regulator of the constitutive expression of HSP73.

Do static or time‐varying magnetic fields in magnetic resonance imaging (3.0 T) alter protein–gene expression?—A study on human embryonic lung fibroblasts

To evaluate the influence of magnetic resonance imaging (MRI) on gene expression in embryonic human lung fibroblasts (Hel 299). The cells were exposed to the static magnetic field and to a turbo spin-echo sequence of an MR scanner at 3.0 Tesla. An MR group (exposed) and a control group (sham-exposed) were set up using a special MR-compatible incubation system. The exposure time was two hours. Gene expression profiles were studied using a complementary deoxyribonucleic acid (cDNA) microarray containing 498 known genes involved in transcription, intracellular transport, structure/junction/adhesion or extracellular matrix, signaling, host defense, energetics, metabolism, cell shape, and death. No changes in gene expression were found in either group (exposed or sham-exposed cells) at the end of a two-hour exposure for any of the 498 tested protein genes. The results suggest that MRI has no influence on protein-gene expression in eugenic human lung cells.

Mechanistic Exploration of Phthalimide Neovascular Factor 1 Using Network Analysis Tools

Neovascularization is essential for the survival and successful integration of most engineering tissues after implantation in vivo. The objective of this study was to elucidate possible mechanisms of phthalimide neovascular factor 1 (PNF1), a new synthetic small molecule proposed for therapeutic induction of angiogenesis. Complementary deoxyribonucleic acid microarray analysis was used to identify 568 transcripts in human microvascular endothelial cells (HMVECs) that were significantly regulated after 24-h stimulation with 30 muM of PNF1, previously known as SC-3-149. Network analysis tools were used to identify genetic networks of the global biological processes involved in PNF1 stimulation and to describe known molecular and cellular functions that the drug regulated most highly. Examination of the most significantly perturbed networks identified gene products associated with transforming growth factor-beta (TGF-beta), which has many known effects on angiogenesis, and related signal transduction pathways. These include molecules integral to the thrombospondin, plasminogen, fibroblast growth factor, epidermal growth factor, ephrin, Rho, and Ras signaling pathways that are essential to endothelial function. Moreover, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) of select genes showed significant increases in TGF-beta-associated receptors endoglin and beta glycan. These experiments provide important insight into the pro-angiogenic mechanism of PNF1, namely, TGF-beta-associated signaling pathways, and may ultimately offer new molecular targets for directed drug discovery.

Downregulation of Extracellular Matrix-Related Gene Clusters during Osteogenic Differentiation of Human Bone Marrow- and Adipose Tissue-Derived Stromal Cells

Bone marrow- and adipose tissue-derived stromal cells (BMSCs and ASCs, respectively) exhibit a similar capacity for osteogenic differentiation in vitro, but it is unclear whether they share a common differentiation process, because they originate from different tissues. The aim of this study was to explore BMSC and ASC osteogenic differentiation by focusing on the expression of extracellular matrix-related genes (ECMGs), which play a crucial role in osteogenesis and bone tissue regeneration in vivo. We characterized the gene expression profiles of BMSCs and ASCs using a custom complementary deoxyribonucleic acid microarray containing 55 ECMGs. Undifferentiated BMSCs and ASCs actively expressed a wide range of ECMGs. Once BMSCs and ASCs were placed in an osteogenic differentiation medium, 24 and 17 ECMGs, respectively, underwent considerable downregulation over the course of the culture period. The remaining genes were maintained at a similar expression level to corresponding uninduced cell cultures. Although the suppression phenomenon was consistent irrespective of stromal cell origin, collagen (COL)2A1, COL6A1, COL9A1, parathyroid hormone receptor, integrin (INT)-beta3, and TenascinX genes were only downregulated in osteogenic BMSCs, whereas COL1A2, COL3A1, COL4A1, COL5A2, COL15A1, osteopontin, osteonectin, and INT-beta1 genes were only downregulated in osteogenic ASCs. During this time period, cell viability was sustained, suggesting that the observed downregulation did not occur by selection and elimination of unfit cells from the whole cell population. These data suggest that osteogenically differentiating BMSCs and ASCs transition away from a diverse gene expression pattern, reflecting their multipotency toward a configuration specifically meeting the requirements of the target lineage. This change may serve to normalize gene expression in mixed populations of stem cells derived from different tissues.

Gene expression profile in squamous cell carcinoma of the urinary bladder using complementary deoxyribonucleic acid microarray

To date, molecular evidence studies for bladder cancer, using the microarray technology, are focusing on the transitional cell carcinoma, however, similar fingerprinting studies have rarely been performed on the other molecular phenotypes of bladder cancer, squamous cell carcinoma (SCC). This study was conducted to monitor the gene expression profiles for bilharzial-related SCC of the bladder to be able to compare its data with transitional cell carcinoma microarray data. A total of 17 paired bilharzial urinary bladder SCC specimens and their corresponding normal urothelium were analyzed using the complementary deoxyribonucleic acid microarray hybridization approach to study the molecular basis of the development of SCC of the urinary bladder. Validation of the microarray results was performed using the Northern blotting technique. After supervised analysis of the microarray data, there was at least a 3-fold difference in the expression between SCC of the bladder and normal urothelium in 82 genes. A total of 38 genes were up-regulated in SCC of the bladder, including matrix degradation-related genes, growth factors, different oncogenes, and immunology related genes. Conversely, 44 genes were down-regulated in SCC of the bladder, including integrins, laminins, cadherins, nonmetastatic cell 1 (NM23) and apoptosis-related genes. Our findings can explain the aggressive behavior of SCC of the bladder. Such gene profiling studies will add to our understanding of the mechanisms of carcinogenesis, and may also improve our ability to diagnose and treat bladder cancer.

Gene Expression Profiling Reveals Platelet-Derived Growth Factor Receptor Alpha as a Target of Cell Contact-Dependent Gene Regulation in an Endothelial Cell–Osteoblast Co-culture Model

Angiogenesis plays an important role in bone development, repair, and remodelling. Neovascularization is furthermore a crucial step in bone tissue engineering because implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of the engineered tissue. We have previously described a co-cultivation system of human primary osteoblasts and human primary endothelial cells that was developed to improve neovascularization in bone tissue-engineering applications. In our present study, we have performed complementary deoxyribonucleic acid microarray analysis to analyze putative changes in osteoblastic gene expression upon co-cultivation of osteoblasts and endothelial cells. Transcriptional profiling revealed upregulation of 79 genes and downregulation of 62 genes in osteoblasts after co-cultivation with endothelial cells. To verify the microarray data, quantitative real-time reverse transcriptase polymerase chain reaction was carried out on selected genes. The expression of the platelet-derived growth factor receptor alpha gene in osteoblasts was analyzed in more detail, revealing that a cell contact-dependent mechanism, and not paracrine-acting diffusible factors, mediates the downregulation of this receptor in osteoblasts upon co-cultivation with endothelial cells. In summary, the data demonstrate complex gene-regulation mechanisms between endothelial cells and osteoblasts that are likely to play a role in bone morphogenesis.

Differences in gene expression between noninvasive and invasive transitional cell carcinoma of the human bladder using complementary deoxyribonucleic acid microarray: Preliminary results

This study was performed to identify differences in gene expression between superficial noninvasive and invasive transitional cell carcinoma (TCC) of the bladder in human beings. We used complementary deoxyribonucleic acid microarrays containing 14,551 different genes to analyze gene expression among 6 cases of superficial and 6 cases of invasive TCC of the bladder to identify differences in gene expression, which might explain differences in the biology and clinical outcomes of these histologic subtypes of TCC. Quantitative real-time polymerase chain reaction was performed for selected genes to validate the microarray data. Significant up-regulation of 40 genes was associated with cases of superficial noninvasive, but not in invasive, TCC of the urinary bladder. This effect included genes involved in epithelial cell dedifferentiation and keratinization, as well as genes related to cell cycle, cell adhesion, transcription, and apoptosis. Conversely, significant up-regulation of 34 genes was associated with cases of invasive TCC, but not in superficial TCC, including genes related to extracellular matrix degradation, immune responses, cell cycling, and angiogenesis. This study shows the usefulness of complementary deoxyribonucleic acid microarray technology for identifying differences in gene expression among different histotypes of bladder cancer.

Micro-array analysis of the effect of post-transurethral bladder tumor resection urine on transforming growth factor-β1 dependent gene expression in transitional cell carcinoma ☆

Introduction and Objectives Prior studies have shown that bladder trauma occurring during transurethral bladder tumor resection increases urinary levels of the cytokine transforming growth factor (TGF)-β1. This study used complementary deoxyribonucleic acid micro-array technology to identify additional genes in human transitional cell carcinoma (TCC), whose expression is altered as a consequence of increased urinary levels of TGF-β1. Methods The human TCC line 253J was cultured in standard media, or media spiked with either 10% post-transurethral bladder tumor resection urine (PTU), or PTU and anti-TGF-β1 neutralizing antibody. Messenger ribonucleic acid from these conditions, together with messenger ribonucleic acid from stably transfected 253J cells over-expressing TGF-β1, was hybridized with ATLAS® micro-array membranes (Clontech, Palo Alto, CA) containing 588 human genes. Hybridization signal intensity was quantified using phospho-imaging. An analytic strategy based on the variance in the signal intensity ratio of specific housekeeping genes in control and experimental comparisons was used to identify significant changes in gene expression. Reverse transcriptase polymerase chain reaction of target genes was used to confirm gene over-expression and TGF-β1 responsiveness. Results Seven genes were identified on micro-array: v-RAF-1, colony stimulating factor-1 receptor, v-FGR, insulin growth factor-1 receptor, epidermal growth factor receptor, α5 integrin, and interferon receptor-1. Reverse transcriptase polymerase chain reaction confirmed over-expression in the autocrine TGF-β1 producing cell line and increased expression in response to exogenous TGF-β1. Conclusions TGF-β1 in PTU alters the expression of multiple genes in human TCC in vitro. The impact of these changes on the biologic phenotype of the malignant cell and the efficacy of adjuvant therapies requires further evaluation.

New Data Analysis and Mining Approaches Identify Unique Proteome and Transcriptome Markers of Susceptibility to Autoimmune Diabetes

Non-obese diabetic (NOD) mice spontaneously develop autoimmunity to the insulin producing beta cells leading to insulin-dependent diabetes. In this study we developed and used new data analysis and mining approaches on combined proteome and transcriptome (molecular phenotype) data to define pathways affected by abnormalities in peripheral leukocytes of young NOD female mice. Cells were collected before mice show signs of autoimmunity (age, 2-4 weeks). We extracted both protein and RNA from NOD and C57BL/6 control mice to conduct both proteome analysis by two-dimensional gel electrophoresis and transcriptome analysis on Affymetrix expression arrays. We developed a new approach to analyze the two-dimensional gel proteome data that included two-way analysis of variance, cluster analysis, and principal component analysis. Lists of differentially expressed proteins and transcripts were subjected to pathway analysis using a commercial service. From the list of 24 proteins differentially expressed between strains we identified two highly significant and interconnected networks centered around oncogenes (Myc and Mycn) and apoptosis-related genes (Bcl2 and Casp3). The 273 genes with significant strain differences in RNA expression levels created six interconnected networks with a significant over-representation of genes related to cancer, cell cycle, and cell death. They contained many of the same genes found in the proteome networks (including Myc and Mycn). The combination of the eight, highly significant networks created one large network of 272 genes of which 82 had differential expression between strains either at the protein or the RNA level. We conclude that new proteome data analysis strategies and combined information from proteome and transcriptome can enhance the insights gained from either type of data alone. The overall systems biology of prediabetic NOD mice points toward abnormalities in regulation of the opposing processes of cell renewal and cell death even before there are any clear signatures of immune system activation.