GeneSpring GX Research Articles

Underexpression of miR-224 in methotrexate resistant human colon cancer cells

MicroRNAs (miRNAs) are small non-coding RNAs involved in RNA silencing that play a role in many biological processes. They are involved in the development of many diseases, including cancer. Extensive experimental data show that they play a role in the pathogenesis of cancer as well as the development of drug resistance during treatments. The aim of this work was to detect differentially expressed miRNAs in MTX-resistant cells. Thus, miRNA microarrays of sensitive and MTX-resistant HT29 colon cancer cells were performed. The results were analyzed using the GeneSpring GX11.5 software. Differentially expressed miRNAs in resistant cells were identified and miR-224, which was one of the most differentially expressed miRNAs and with high raw signal values, was selected for further studies. The underexpression of miR-224 was also observed in CaCo-2 and K562 cells resistant to MTX. Putative targets were predicted using TargetScan 5.1 software and integrated with the data from expression microarrays previously performed. This approach allowed us to identify miR-224 targets that were differentially expressed more than 2-fold in resistant cells. Among them CDS2, DCP2, HSPC159, MYST3 and SLC4A4 were validated at the mRNA level by qRT-PCR. Functional assays using an anti-miR against miR-224 desensitized the cells towards MTX, mimicking the resistant phenotype. On the other hand, siRNA treatment against SLC4A4 or incubation of Poly Purine Reverse Hoogsteen (PPRH) hairpins against CDS2 or HSPC159 increased sensitivity to MTX. These results revealed a role for miR-224 and its targets in MTX resistance in HT29 colon cancer cells.

Histone deacetylase inhibitor enhances the anti-tumor effect of gemcitabine: A special reference to gene-expression microarray analysis

Histone deacetylase (HDAC) is strongly associated with epigenetic regulation and carcinogenesis, and its inhibitors induce the differentiation or apoptosis of cancer cells. Valproic acid (VPA) is one of the clinically available HDAC inhibitors. We investigated the anticancer effects of VPA in combination with gemcitabine (GEM) in the human cholangiocarcinoma cell line HuCCT1, and explored the mechanisms of the anticancer effects using microarray analysis. The anticancer effects of VPA or gemcitabine (GEM), and the effects of VPA combined with GEM, were studied by a cell proliferation assay. A microarray analysis was performed and the genes were picked up using GeneSpring GX11.5, followed by Ingenuity Pathways Analysis (IPA) and determination of gene expression by RT-PCR. GEM (5 nM) and VPA (0.5 mM) reduced proliferation by 23%, which significantly augmented the anticancer effect of GEM alone or VPA alone (P<0.01). Using microarray analysis, 43 genes were identified with the comparison between the GEM group and the GEM plus VPA combination group. Interactions were identified between genes of the 'Cellular Development' network relevant to the differentiation of cancer cells using IPA. Furthermore, GEM combined with VPA up-regulated the HLA-DRA expression compared to the single agents (P<0.01). VPA augmented the effects of GEM by enhancing the gene network mainly including HLA-DRA, possibly through the modification of cancer cell differentiation.

Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells as demonstrated by genome-wide microarray analysis

Platelets contain numerous growth factors essential for wound and fracture healing. We investigated the gene expression in human osteoblast-like cells stimulated with lysed platelets prepared in acidic, neutral, or alkaline buffers. Lysed platelets prepared in buffers at pH 5.4, 7.4, and 7.9, were added after neutralization to hFOB 1.19 cells. Genome-wide microarray analysis was performed using the Affymetrix GeneChip 7G Scanner. Biometric, cluster, and pathway analyses were performed with GeneSpring GX. Biometric analyses demonstrated that 53 genes were differentially regulated (p ≤ 0.005, ≥2-fold increase). Pathway analysis revealed 10 significant pathways of which eight are common ones regulating bone formation and cancer growth. Eleven genes were selected for quantitative real-time polymerase chain reaction (PCR) based on the microarray analysis of the lysed platelets prepared in the pH 5.4 experiments. In conclusion, acidic preparations of lysed platelet concentrates release factors essential for cell proliferation and particularly cell metabolism under hypoxic conditions. The genetic response from these factors was dominated by genes associated with the same pathways observed in bone formation and cancer growth. Activation of TGF-β in the acidic preparation could be a stimulatory key factor of cell proliferation. These results support the hypothesis that acidification of platelets modifies the stimulatory response of mesenchymal cells in vitro, which is analogous with the observed milieu of a low pH present in wound and fracture sites, as well as in growing tumors.

MicroRNA-145 Regulates Human Corneal Epithelial Differentiation

BackgroundEpigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.Methodology/Principal FindingsHuman LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.Conclusion/SignificanceWe found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.

Abstract 5011: Difference in expression of STAT3 and STAT5b in Swedish and Egyptian patients with activated b-cell subtype of diffuse large b-cell lymphoma

Abstract Diffuse Large B-cell lymphoma (DLBCL), the most common type of lymphoma in adults, accounts for approximately 30 to 40% of newly diagnosed lymphomas. Using gene expression profiling, DLBCL can be divided into three immunologically indistinguishable subtypes: the germinal center B-cell like (GBC), the activated B-cell like (ABC) and the primary mediastinal B-cell lymphoma (PMBL) subtype. The aim of the study was to compare gene expression profiles of Swedish and Egyptian ABC DLBCL patients. Formalin fixed and paraffin embedded material of 67 Egyptian and 15 Swedish ABC DLBCL patients were used in the study. Affymetrix gene expression platform was used for profiling and the analysis was carried out using GeneSpring GX software. Quantitative PCR (qPCR) and confocal microscopy was used to verify the findings. The GeneSpring GX analysis showed that 3247 genes were at least 2 fold up- or downregulated comparing the two patient groups. Among these were 1827 genes upregulated and 1420 genes downregulated. Gene expression analysis was verified using qPCR showing that STAT3 was upregulated with a fold change of 3.1 in the Swedish patients and that STAT5b was upregulated with a fold change of 1.3 in the Egyptian patients. Confocal microscopy showed protein activity of STAT3 and 5b in the nucleus of Swedish and Egyptian patients respectively using monoclonal antibodies. This is the first study that reveals diversity in expression of STATs in different populations of ABC DLBCL patients. STAT3 and 5b are known to have different pathways of activation and targeted disruption has shown their functional differences. Using gene expression profiling, we revealed different expression levels of STAT3 and STAT5b in Swedish and Egyptian ABC DLBCL patients. Since the populations of Sweden and Egypt are exposed to different infectious agents, the results of the present study further raise the question concerning viral etiology in ABC-DLBCL possibly due to repeated infections and chronic inflammation. In general, these findings give additional evidence to the importance of signaling pathways involving STATs in ABC DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5011. doi:10.1158/1538-7445.AM2011-5011

Abstract 4867: Whole transcript expression microarray profiling of normal colon and adenocarcinoma total RNA

Abstract Whole transcript expression profiling by microarray analysis is an important tool for understanding biological mechanisms and development, classifying tissue and tumor types, and identifying indicators for diagnosis and prognosis. This analysis measures the alternative splicing of exons from a given RNA transcript leading to the translation of a variety of proteins from a single RNA transcript. To address the need for whole transcriptome expression profiling from low quantities of total RNA, we have developed an exon expression workflow which includes custom and catalog human, mouse, and rat exon microarrays, a whole transcript labeling method, and analysis software. This fast and simple microarray-based method employs a modified linear amplification procedure to generate Cy-labeled cRNA representing the whole transcript. The labeling method, available in the Low Input Quick Amp Labeling WT Kit (LIQA WT), employs a mixture of oligo dT- and random nucleotide-based T7 promoter primers (WT Primer Mix) resulting in high cRNA yields and specific activities from low RNA inputs. The probes on the human, mouse, and rat SurePrint G3 exon microarrays were designed from the high quality content of the public databases, primarily RefSeq and Ensembl. Exon array results are analyzed for gene and exon-level expression using GeneSpring GX 11.5 software. The exon gene expression workflow allowed whole transcript gene expression profile comparisons to be made in fewer than two days. Comparisons of probe signals from technical replicate samples demonstrated high reproducibility with a wide dynamic range and comparable signals across a broad range of input amounts. High correlations were demonstrated in platform comparisons to both qRT-PCR and RNA-Seq. This new workflow was used to detect alternative splicing of exons between cancerous and normal cells resulting in gene and exon expression profiles consistent with the current literature. These results demonstrate the value of the new exon expression workflow in identifying known exons, alternative start and stop sites, mutually exclusive exons, and cassette exons in cancer research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4867. doi:10.1158/1538-7445.AM2011-4867

Abstract 4121: Folate pathway in malignant pleural mesothelioma (MPM): Novel therapeutic opportunities due to folate receptor alpha overexpression

Abstract Background. The folate pathway is active in several cancers including malignant pleural mesothelioma (MPM). Membrane receptors folate receptor alpha (FRα), reduced folate carrier 1 (RFC1), proton coupled folate transporter (PCFT) and the enzyme thymidylate synthase (TS) regulate intra-cellular uptake of folate molecules and antifolate drugs. Due to the documented overexpression of folate markers in MPM they arise as potential targets for novel specific therapies with humanized monoclonal anti-FRα antibody and also to antifolate chemotherapy with pemetrexed. Material and Methods. We studied 79 surgically resected MPM (43 epithelioid, 28 biphasic and 13 sarcomatoid histology types). Protein expression of FRα, RFC1, PCFT and TS was examined by immunohistochemistry (IHC) in FFPE tissues placed in tissue microarray platform and results correlated to clinical-pathologic characteristics. Gene expression and DNA copy number changes of the FRα gene (FOLR1) were examined in 53 mesothelioma tumors using Affymetrix U133 Plus 2.0 microarray's and Illumina HumanOmni1-Quad v1.0 chips, respectively. The expression microarray data was analyzed in the GeneSpring GX 11 software (Agilent technologies Inc.) while DNA copy number changes were detected using the Nexus 5.0 software (BioDiscovery Inc.). Results. MPM frequently showed protein over-expression of FRα, RFC1 and PCFT. Nuclear TS expression positively correlated (P&amp;lt;0.043) with cytoplasmic TS and membrane RFC1. Patients with T1/T2 tumor had significantly (P=0.038) higher levels of membrane FRα, compared to patients with T3/T4 tumors. Epitheloid MPM had the highest level of cytoplasmic PCFT (P=0.005) among the three histological types, while patients with sarcomatoid histology had the lowest. In the multivariate analysis, with adjustment of gender, N and M stage, Patients who expressed any nuclear TS (score &amp;gt;0) had a lower hazard ratio than those who did not (HR=0.309; 95%CI 0.159, 0.601; P=0.0005). Patients with any level (score &amp;gt;0) of cytoplasmic PCFT had lower hazard ratio than those with absence of the marker (HR=0.539; 95%CI 0.306, 0.952; P=0.0332). Our gene expression data analysis on 53 mesothelioma tumors showed greater than 2-fold increase in the FOLR1 and 3-fold increase in the TYMS messenger RNA levels compared to paired normal tissue. Additionally, at least 7 of these tumors show increased gene copy number at the FOLR1 locus (Ch11q 13.4). Conclusions. Folate membrane transporters (FRα, RFC1, PCFT) and the enzyme TS are frequently over expressed in MPM tumor tissues, and PCFT and TS are associated with tumors’ clinico-pathological features. The high level of expression of FRα in MPM supports the rational use of potential utilization of humanized monoclonal anti-FRα antibody and also standard antifolate chemotherapy in this disease. Supported by grant US DoD W81XWH-07-1-0306, ASCO CDA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4121. doi:10.1158/1538-7445.AM2011-4121

Abstract 4031: CNG c-myc in mesothelioma

Abstract Background Malignant pleural mesothelioma (MPM) is a deadly disease with poor prognosis, few treatment options, and an increasing incidence worldwide. There is, therefore, a great need to identify new therapeutic targets and develop more effective therapies for patients with MPM. To better characterize the molecular changes occurring in MPM, we determined genetic and proteomic abnormalities in MPM cell lines and correlated them with those found in MPM tumor tissue specimens. Methods We performed SNP/copy number analysis using Affymetrix SNP 6.0 chips, messenger RNA (mRNA) analysis using Affymetrix U133 plus 2.0 chips, and Reverse Protein Phase Array (RPPA) analysis in 4 mesothelioma cell lines (H28, MSTO-211H, H2052 and H2452) along with the control normal cell line HCT-4012, for comparison. DNA copy number microarray data were analyzed using the Nexus 5.0 software (BioDiscovery Inc.), while mRNA expression array data were analyzed using the GeneSpring GX 11 software (Agilent technologies, Inc.). Additionally, we evaluated copy number gain (CNG) of the c-myc gene and expression of c-myc protein in the mesothelioma cell lines and control cell line by fluorescent in situ hybridization (FISH) and Western blot, respectively. Furthermore, we investigated CGN in MPM tumor tissue specimens by performing FISH using the c-myc probe on tissue microarrays (TMAs) containing 80 MPM samples from different histological subtypes (41 epithelioid, 27 biphasic, 12 sarcomatoid). Results DNA copy number analysis using the Affymetrix chip revealed CNG or amplification of the c-myc oncogenic locus in 3 out of 4 mesothelioma cell lines compared to the control HCT-4012 cell line. In concordance, the mRNA microarray data also showed increased number of transcripts from this locus; more importantly, the RPPA array data showed increased c-myc protein expression in these cell lines compared to control. These results were confirmed by FISH and Western blot analysis, which showed CNG or amplification at this locus and increased levels of the c-myc protein in the mesothelioma cell lines. FISH analysis of TMAs also revealed a relatively high frequency (21%) of CNG (≥4 copies in ≥70% of cells) for the c-myc locus in the MPM tumors. Interestingly, this amplification were seen in either epithelioid (10%) or biphasic (11%) histotype, and none were observed in the sarcomatoid cases. Similarly, within the biphasic cases, we observed that the amplification was present only in the epithelioid component, and not in the sarcomatoid component. Conclusion Our findings suggest that CNG of the c-myc locus is characteristic for MPM tumors of the epithelioid histotype or of the epitheloid component of biphasic histotype, and suggest that c-myc CNG could have a role in pathogenesis of this disease; however, further studies are needed to clarify the role of c-myc in MPM development and progression. Supported by grant US DoD W81XWH-07-1-0306. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4031. doi:10.1158/1538-7445.AM2011-4031

Abstract 3676: The use of r-hCG changes the transcriptome profile of nulliparous women carrying BRCA1 mutation

Abstract Nulliparity and inheritance of BRCA1 or BRCA2 mutations are conditions associated with a greater risk of developing breast cancer. The knowledge that early parity reduces a woman's lifetime cancer risk and the demonstration in preclinical studies that the protective effect of pregnancy is mediated by the differentiation of the breast, which is manifested as permanent changes in the genomic/transcriptomic profile of this organ, led us to hypothesize that the transcriptomic profile of breast tissue of nulliparous BRCA1 mutation carriers would revert from high risk to lower risk after a short treatment with recombinant human chorionic gonadotropin (r-hCG). For this purpose we designed a pilot study for determining whether treatment of sexually mature, from 20 to 40 years of age, nulliparous women carriers of BRCA1 mutations with the r-hCG changes their breast epithelium's genomic profile to one similar to that identified in cancer-free postmenopausal women with a history of full term pregnancy. After signing an informed consent form, eligible candidates received 250 micrograms r-hCG applied as a subcutaneous injection 3 times a week for 12 weeks. Before initiation of treatment (T0), at the end (T12), and 24 weeks post-treatment (T24) a breast core needle biopsy (CNB) was performed by the study surgeon. In this proof of concept, tissues obtained from two volunteers were divided for histopathological and RNA analyses. Total RNA was extracted, prepared for hybridization using Two-Cycle Target Labeling and Control Reagents kits from Affymetrix, and hybridization to Affymetrix Human Genome U133 Plus 2.0 chips. The chips were analyzed using GeneSpring GX v11.0 software (Agilent Technologies). Comparison of gene expression between T0 and T24 revealed 425 probes (254 up- and 171 down-regulated), representing 349 unique differentially expressed genes (p-value&amp;lt; 0.01). Gene Ontology (GO) analysis revealed that enriched biological processes (p&amp;lt;0.1) were related mainly to regulation of gene expression and transcription, such as chromatin-mediated maintenance of transcription (GO:0048096); regulation of DNA-dependent transcription (GO:0006355); positive regulation of epigenetic gene expression (GO:0045815), and RNA metabolic process (GO:0051252). Regulation of genes related to splicing factors (SF3A1, SFRS12IP1); RNA binding motifs proteins (RBM12, RBM12B, RBM16, RBM22), and methyltransferase like (METTL9, METTL14) was also observed. The changes in the breast's transcriptomic pattern induced by the r-hCG treatment were similar to those that have been detected in cancer-free postmenopausal parous women. These observations led us to conclude that this short term hormonal treatment activates biological processes that could confer a degree of protection from developing breast cancer similar to that induced by pregnancy. (Study supported by NCI grant R21 CA124522). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3676. doi:10.1158/1538-7445.AM2011-3676

Abstract 167: Identification of miR-34a as a prognostic biomarker of Ewing sarcoma family of tumors

Abstract Clinical outcomes of Ewing sarcoma family of tumors (ESFT) were improved after the introduction of multimodal therapies including intensive chemotherapy. However current therapeutic approaches still leave us the problems including short-term toxicities of chemotherapy and serious late adverse effects; second malignancy and fatal cardiac failure even for long survivors. Therefore, we are in the urgent need to develop the novel biomarkers for an appropriate stratification of patients at diagnosis to establish more effective and less invasive therapy for all ESFT patients. MicroRNAs (miRNAs) are newly-recognized small non gene-coding RNA family that regulate gene expression through translational suppression. More recently, they have been increasingly accepted as playing crucial roles in pathogenesis of most human malignancy as well as normal cell differentiation. However, little is known regarding to the possible role of miRNAs on sarcoma including ESFT. Here we focus on the whole profiling of the miRNA expressions in ESFT for the aim to elucidate the novel miRNA biomarkers in the treatment of ESFT. Randomly selected 34 primary biopsy samples of ESFT have been analyzed for complete miRNA expression profiles. All of the patients were treated at Rizzoli Institute from 1991 to 2008 with multimodal therapies. Microarray and statistical analyses were carried out using Agilent's miRNA microarray kit (v2) and GeneSpring GX analysis software, respectively. To identify miRNAs associated with the prognosis of ESFT patients we first compared the expression profiles of 21 patients who relapsed within 5 years with 13 patients who remained free of disease throughout the follow-up. As a result, we identified 16 miRNAs differentially expressed between the two groups (fold change &amp;gt; 1.5, p&amp;lt;0.1). We have also performed univariate Cox regression analysis to elucidate the miRNAs, whose expression level are related to the length of event-free survival (EFS) and/or overall survival (OS) and found out 15 miRNAs and 32 miRNAs that significantly correlated with EFS and OS, respectively. We then validated these data with quantitative real time PCR using extended series of 49 primary biopsy samples. After these validation, we have confirmed that low expressions of miR-34a were strongly associated with worse prognosis (P&amp;lt;0.001, Logrank test) and remained most significant biomarker after multivariate analyses. Moreover expression levels of miR-34a using real time PCR were highly correlated with the level of corresponding microarray data (Pearson correlation test: p&amp;lt;0.0001). Over-expression of miR-34a inhibited the colony formation in the soft agar and enhanced chemosensitivities of ESFT cell lines. In conclusion, we have identified miR-34a as a powerful biomarker to distinguish the prognosis of ESFT patients through the whole miRNA profiling analyses (grants from EU project Eurobonet and Italian Association for Cancer Research). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 167. doi:10.1158/1538-7445.AM2011-167

Gene expression profiles in rat mesenteric lymph nodes upon supplementation with Conjugated Linoleic Acid during gestation and suckling

BackgroundDiet plays a role on the development of the immune system, and polyunsaturated fatty acids can modulate the expression of a variety of genes. Human milk contains conjugated linoleic acid (CLA), a fatty acid that seems to contribute to immune development. Indeed, recent studies carried out in our group in suckling animals have shown that the immune function is enhanced after feeding them with an 80:20 isomer mix composed of c9,t11 and t10,c12 CLA. However, little work has been done on the effects of CLA on gene expression, and even less regarding immune system development in early life.ResultsThe expression profile of mesenteric lymph nodes from animals supplemented with CLA during gestation and suckling through dam's milk (Group A) or by oral gavage (Group B), supplemented just during suckling (Group C) and control animals (Group D) was determined with the aid of the specific GeneChip® Rat Genome 230 2.0 (Affymettrix). Bioinformatics analyses were performed using the GeneSpring GX software package v10.0.2 and lead to the identification of 89 genes differentially expressed in all three dietary approaches. Generation of a biological association network evidenced several genes, such as connective tissue growth factor (Ctgf), tissue inhibitor of metalloproteinase 1 (Timp1), galanin (Gal), synaptotagmin 1 (Syt1), growth factor receptor bound protein 2 (Grb2), actin gamma 2 (Actg2) and smooth muscle alpha actin (Acta2), as highly interconnected nodes of the resulting network. Gene underexpression was confirmed by Real-Time RT-PCR.ConclusionsCtgf, Timp1, Gal and Syt1, among others, are genes modulated by CLA supplementation that may have a role on mucosal immune responses in early life.

Effects of organic and inorganic dietary selenium supplementation on gene expression profiles in oviduct tissue from broiler-breeder hens

Selenium (Se) is an essential component of at least 25 selenoproteins involved in a multitude of physiological functions, including reproduction. However, relatively little is known about the mechanisms by which Se exerts its physiological effects in reproductive tissue. The objective of this study was to compare the effect of long-term inorganic Se (sodium selenite, SS) and organic yeast-derived Se (Sel-Plex ®, SP) supplementations on tissue Se content and gene expression patterns in the oviduct of broiler-breeder hens. Hens were randomly assigned at 6 weeks of age to one of the three treatments: basal semi-purified diet (control), basal diet + 0.3 ppm Se as SP or basal diet + 0.3 ppm Se as SS. At 49 weeks, oviduct tissue from hens randomly selected from each treatment ( n = 7) was analyzed for Se content and gene expression profiles using the Affymetrix Chicken genome array. Gene expression data were evaluated using GeneSpring GX 10.0 (Silicon Genetics, Redwood, CA) and Ingenuity Pathways Analysis software (Ingenuity Systems, Redwood City, CA). Oviduct Se concentration was greater with Se supplementation compared with the control ( P ≤ 0.05) but did not differ between SS- and SP-supplemented groups. Gene expression analysis revealed that the quantity of gene transcripts associated with energy production and protein translation were greater in the oviduct with SP but not SS supplementation. Targets up-regulated by SP, but not SS, included genes encoding several subunits of the mitochondrial respiratory complexes, ubiquinone production and ribosomal subunits. SS hens showed a decrease in transcripts of genes involved in respiratory complexes, ATP synthesis and protein translation and metabolism in oviduct relative to control hens. In this study, although tissue Se concentrations did not differ between hens fed SS- and SP-supplemented diets, expression patterns of genes involved in energy production and protein synthesis pathways differed between treatments. These variations may partially explain the differences in reproductive performance reported in hens fed different forms of Se.

Progesterone Resistance in PCOS Endometrium: A Microarray Analysis in Clomiphene Citrate-Treated and Artificial Menstrual Cycles

Polycystic ovary syndrome (PCOS), the most common endocrinopathy of reproductive-aged women, is characterized by ovulatory dysfunction and hyperandrogenism. The aim was to compare gene expression between endometrial samples of normal fertile controls and women with PCOS. We conducted a case control study at university teaching hospitals. Normal fertile controls and women with PCOS participated in the study. Endometrial samples were obtained from normal fertile controls and from women with PCOS, either induced to ovulate with clomiphene citrate or from a modeled secretory phase using daily administration of progesterone. Total RNA was isolated from samples and processed for array hybridization with Affymetrix HG U133 Plus 2 arrays. Data were analyzed using GeneSpring GX11 and Ingenuity Pathways Analysis. Selected gene expression differences were validated using RT-PCR and/or immunohistochemistry in separately obtained PCOS and normal endometrium. ANOVA analysis revealed 5160 significantly different genes among the three conditions. Of these, 466 were differentially regulated between fertile controls and PCOS. Progesterone-regulated genes, including mitogen-inducible gene 6 (MIG6), leukemia inhibitory factor (LIF), GRB2-associated binding protein 1 (GAB1), S100P, and claudin-4 were significantly lower in PCOS endometrium; whereas cell proliferation genes, such as Anillin and cyclin B1, were up-regulated. Differences in gene expression provide evidence of progesterone resistance in midsecretory PCOS endometrium, independent of clomiphene citrate and corresponding to the observed phenotypes of hyperplasia, cancer, and poor reproductive outcomes in this group of women.

Cyclin D1 Is a Selective Modifier of Androgen-dependent Signaling and Androgen Receptor Function

D-type cyclins regulate cellular outcomes in part through cyclin-dependent, kinase-independent mechanisms that modify transcription factor action, and recent in vivo studies showed that cyclin D1 associates with a large number of transcriptional regulators in cells of the retina and breast. Given the frequency of cyclin D1 alterations in cancer, it is imperative to delineate the molecular mechanisms by which cyclin D1 controls key transcription factor networks in human disease. Prostate cancer was used as a paradigm because this tumor type is reliant at all stages of the disease on androgen receptor (AR) signaling, and cyclin D1 has been shown to negatively modulate AR-dependent expression of prostate-specific antigen (KLK3/PSA). Strategies were employed to control cyclin D1 expression under conditions of hormone depletion, and the effect of cyclin D1 on subsequent androgen-dependent gene expression was determined using unbiased gene expression profiling. Modulating cyclin D1 conferred widespread effects on androgen signaling and revealed cyclin D1 to be a selective effector of hormone action. A subset of androgen-induced target genes, known to be directly regulated by AR, was strongly suppressed by cyclin D1. Analyses of AR occupancy at target gene regulatory loci of clinical relevance demonstrated that cyclin D1 limits AR residence after hormone stimulation. Together, these findings reveal a new function for cyclin D1 in controlling hormone-dependent transcriptional outcomes and demonstrate a pervasive role for cyclin D1 in regulating transcription factor dynamics.

A microarray based expression profiling of paclitaxel and vincristine resistant MCF-7 cells

Resistance to the broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. Drug resistant sublines to paclitaxel (MCF-7/Pac) and vincristine (MCF-7/Vinc) that were developed from sensitive MCF-7 cells (MCF-7/S) were used. cDNA microarray analysis was performed for the RNA samples of sensitive and resistant cells in duplicate experiments. GeneSpring GX 7.3.1 Software was used in data analysis. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of the paclitaxel and vincristine drug resistance. Additionally the upregulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant downregulation of apoptotic genes (i.e. PDCD2/4/6/8) and upregulation of some cell cycle regulatory genes ( CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Drug resistant cancer cells exhibit different gene expression patterns depending on drug treatment, and each drug resistance phenotype is probably genetically different. Further functional studies are needed to demonstrate the complete set of genes contributing to the drug resistance phenotype in breast cancer cells.

Unique Gene Expression Patterns in Hematopoietic Stem Cells from Various Sources

Hematopoietic stem cells are involved in the production of blood cells. These cells are derived from various origins. In this study, we examined, using microarray software GeneSpring GX, whether HSCs derived from four different origins i.e., cord blood, liver, bone marrow and peripheral blood, exhibit unique gene expression pattern. A total of 23 individual normal hematopoietic cell expression profiles were identified in NCBI's GEO database -10 normal CD34+ bone marrow sample, 4 normal CD34+ liver sample, 5 normal CD34+ cord blood sample and 4 normal CD34+ peripheral blood sample. These studies were performed using U133A array platform. The data analysis was done by GeneSpring GX Microarray software 7.3 version. Our results indicated that HSCs derived from various origins exhibited unique gene expression patterns. We also determined the specific biological function of HSCs using upregulated genes that are uniquely expressed in each origin. We observed the following results - cord blood HSCs play an important role in development of embryos; Liver HSCs play an important role in apoptosis, angiogenesis and neural transdifferentiation; Bone marrow HSCs play an important role in cell proliferation, cell migration and cell retention and peripheral blood HSCs play an important role in nucleosome assembly. These HSCs genes present in peripheral blood, when altered, get involved in Systemic Lupus Erythematosus Pathway.

Differential expression of elastin assembly genes in patients with Stanford Type A aortic dissection using microarray analysis

Pathologic studies have demonstrated that aortic dissection is initiated by an intimal tear, followed by the rapid growth of an intramural hematoma that dissects the media and is characterized by elastin degradation. Genetic extracellular matrix abnormalities and proteinases may be the predisposing factors in aortic dissection, but little is known about the role of elastic fiber assembly. Fibulin-1 is an extracellular protein that is expressed in the vascular basement membrane. It regulates elastic fiber assembly and hence provides integrity in aortic structure. This study investigates the expression profiles of genes responsible for the elastolysis in the dissected human aorta, especially those coding fibulin-1, matrix metalloproteinase-9 (MMP-9), and elastin. Intraoperative aortic samples were obtained from Chinese patients with Stanford Type A aortic dissection. Both the ascending dissected aortas (primary tear) and the adjacent intact aortas were collected for comparison. Control aortic tissues were obtained from healthy organ donors. The gene profile study was determined by the Affymetrix HG-U133A GeneChip (Affymetrix, Santa Clara, Calif) and analyzed by GeneSpring GX11.0 (Agilent Technologies Inc, Palo Alto, Calif). Only the genes displaying a net signal intensity two-fold higher than the mean background were used for analysis. To evaluate elastin expression, aortic sections were stained with Movat pentachrome stain. Fibulin-1, MMP-9, and elastin mRNA and protein expression were further confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblotting, respectively. Eight male Chinese aortic dissection patients (mean age, 45.8 years) and eight gender- and age-matched organ donors were recruited for the study. On the Affymetrix platform, 2,250 of 22,283 genes (10.1%) were detectable. The dissected and adjacent macroscopically intact aorta displayed similar gene expression patterns. In contrast, 11.2% (252) of the detectable genes were differentially expressed in the dissected and control aortas. Of these, 102 genes were upregulated, and 150 genes were downregulated. Based on the gene ontology, genes that code for extracellular matrix protein components and regulating elastic fiber assembly, like fibulin-1 and elastin, were downregulated, while enzymes like MMP-9 and MMP-11 that degrade matrix proteins were upregulated in dissected aortas. RT-PCR and Western blot results further validated the results. Our gender- and age-matched study demonstrated that the alternated genes in the elastin assembly of dissected aortas may predispose structural failure in the aorta leading to dissection. However, no significant gene alterations in the adjacent intact and dissected aortas of the same patient can be found. Therefore, the genetic changes found in the dissected aortas most likely developed before the dissection starts. The inhibition of the aberrant expression of the fibulin-1 gene and that of the related matrix proteinase may open a new avenue for preventing aortic dissection.

Gene profiling of early and advanced liver disease in chronic hepatitis C patients

Strong impact of hepatitis C virus (HCV) on normal regulation of cellular processes has been reported that could have significant implications for HCV pathogenesis. We aimed to determine the altered cellular processes during HCV infection with particular reference to advanced disease stages. Liver biopsy specimens of chronic hepatitis C patients classified on histological basis as early (fibrosis stage 1-2) or advanced (fibrosis stage 3-4) HCV disease were studied using microarray technology (Affymetrix GeneChip™ System). For comparison, liver specimens from patients with non-viral hepatitis (NV-hepatitis) were also analyzed by microarray. Expression data generated were analyzed using software Genespring GX and Ingenuity Pathway analysis to find the association with biological functions. We further validated the microarray results using quantitative reverse transcriptase-polymerase chain reaction. Data analysis through Genespring software revealed that in advanced HCV (A-HCV) a total of 792 genes are differentially expressed when compared to early HCV (E-HCV) and 417 genes are differentially expressed when compared to NV-hepatitis. Most of these genes are involved in cancer, cellular growth and proliferation, and tissue morphology. Real time (RT) PCR analysis confirmed the differential expression of six of these genes. The results of this study reflect the changes taking place during the transition from early to advanced liver fibrosis, when the liver function becomes impaired and extracellular matrix deposition increases. In addition, it showed altered expression of genes with functions in cancer development, cell growth, proliferation, and cell death that might indicate high risk of cell transformation and hepatocellular carcinoma (HCC) in A-HCV disease patients.

Gene expression profiles in aldosterone-producing adenomas and adjacent adrenal glands

Primary aldosteronism (PA) is the most common form of endocrine hypertension affecting ∼8-10% of hypertensive subjects. Aldosterone production in PA occurs under low-renin conditions, and the mechanisms that maintain the production of aldosterone in PA remain unknown. Objective This study was designed to compare the transcript profiles between aldosterone-producing adenoma (APA) and their adjacent adrenal gland (AAG) from the same adrenal. Total RNA was extracted from ten APA and ten AAG; and subsequently analyzed by microarray and real-time quantitative RT-PCR (qPCR). The microarray data were paired for each APA-AAG, and analyzed by GeneSpring GX 11 with paired t-test and fold change calculations for each transcript. Changes identified by microarray analysis were confirmed by qPCR. Microarray analysis indicated that 14 genes had significantly up-regulated expression in APA compared to AAG. Among the elevated genes were aldosterone synthase (CYP11B2) as well as novel transcription factors, calmodulin-binding proteins, and other genes that have not been previously studied in APA. Selective analysis of 11 steroidogenic enzymes using microarray demonstrated that only CYP11B2 showed a significantly higher transcript level in APA compared to AAG (P<0.001). In contrast, AKR1C3 (17β-hydroxysteroid dehydrogenase type 5), CYP17 (17α-hydroxylase/17,20 lyase), and CYB5 (cytochrome b5) showed significantly lower transcript level in APA (P<0.05). The transcriptome analysis of APA compared with AAG showed several novel genes that are associated with APA phenotype. This gene list provides new candidates for the elucidation of the molecular mechanisms leading to PA.

IDENTIFICATION OF GENDER-SPECIFIC TRANSCRIPTS BY MICROARRAY IN GONAD TISSUE OF LARVAL AND JUVENILE XENOPUS TROPICALIS

Event Abstract Back to Event IDENTIFICATION OF GENDER-SPECIFIC TRANSCRIPTS BY MICROARRAY IN GONAD TISSUE OF LARVAL AND JUVENILE XENOPUS TROPICALIS Jonathan T. Haselman1*, Allen W. Olmstead1 and Sigmund J. Degitz1 1 US Environmental Protection Agency, Mid-Continent Ecology Division, United States Amphibian model species Xenopus tropicalis is currently being utilized by EPA in the development of a standardized in vivo reproductive toxicity assay. Perturbations to the hypothalamic-pituitary-gonadal axis from exposure to endocrine disrupting compounds during larval development have been shown to induce abnormal gonad differentiation resulting in either functional sex-reversal or intersex conditions. In an effort to develop a gene expression-based diagnostic tool we characterized male and female gonadal transcriptomes by microarray. Gonad tissues were harvested from Nieuwkoop and Faber (NF) stage 58 larvae, NF 66 froglets, one week and two week old juveniles. Individual animals were genotyped to confirm genetic sex and gonad RNA was extracted and pooled according to developmental stage and gender. Initial data analyses were performed with Agilent GeneSpring GX 11 and gene enrichment and ontology analyses were carried out with gender-specific gene lists using DAVID Bioinformatics Resources 6.7. Results confirm significant differences (p≤ 0.05 and differential expression ≥ 10-fold) in expression in two week old juveniles between ovary and testis tissues of several enzymes involved in steroid hormone biosynthesis. Other ovary-specific profiles include oocyte-specific proteins and several proteins involved in progesterone mediated oocyte maturation and oocyte meiosis. Additional testis-specific profiles include a Leydig cell-specific peptide and hormone receptors. These data allow for further investigation of molecular interactions responsible for gonad differentiation and will provide candidate genes for use as expression-based biomarkers of gonad maturation. Examination of these expression profiles in gonad tissue influenced by xenobiotic exposure will provide insight into molecular mechanisms and processes leading to amphibian gonadal intersex and functional sex-reversal. This abstract does not necessarily reflect U.S. EPA policy. Acknowledgements The authors would like to thank Hongzu Ren from the Genomics Research Core of US EPA for preparing and analyzing the Affymetrix array chips. The authors would also like to thank Chad Blanksma for the gonad histology processing and images. References Nieuwkoop, P. and Faber, J. (1967). Normal Table of Xenopus laevis. Daudin, North-Holland, Amsterdam Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. Nature Protoc. 2009;4(1):44-57. Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37(1):1-13. Olmstead AW, Lindberg-Livingston A, Degitz SJ. Genotyping sex in the amphibian, Xenopus (Silurana) tropicalis, for endocrine disruptor bioassays. Aquatic Toxicology. 2010;98(1):60-66. Keywords: Gonad differentiation, Intersex, Microarray, Sex reversal, Xenopus tropicalis Conference: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology, Ann Arbor, United States, 11 Jul - 13 Jul, 2011. Presentation Type: Poster Topic: Sex determination and differentiation Citation: Haselman JT, Olmstead AW and Degitz SJ (2011). IDENTIFICATION OF GENDER-SPECIFIC TRANSCRIPTS BY MICROARRAY IN GONAD TISSUE OF LARVAL AND JUVENILE XENOPUS TROPICALIS. Front. Endocrinol. Conference Abstract: ISAREN 2011: 7th International Symposium on Amphibian and Reptilian Endocrinology and Neurobiology. doi: 10.3389/conf.fendo.2011.03.00016 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Mr. Jonathan T Haselman, US Environmental Protection Agency, Mid-Continent Ecology Division, Duluth, MN, 55804, United States, haselman.jon@epa.gov Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract Supplemental Data The Authors in Frontiers Jonathan T Haselman Allen W Olmstead Sigmund J Degitz Google Jonathan T Haselman Allen W Olmstead Sigmund J Degitz Google Scholar Jonathan T Haselman Allen W Olmstead Sigmund J Degitz PubMed Jonathan T Haselman Allen W Olmstead Sigmund J Degitz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.