Unknown,Transcriptomics,Genomics,Proteomics

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Environmental enteric dysfunction includes a broad spectrum of inflammatory responses and epithelial repair processes (RT-PCR)

ABSTRACT: Environmental enteric dysfunction (EED), a chronic diffuse inflammation of the small intestine, is associated with stunting in children in the developing world. The pathobiology of EED is poorly understood because of the lack of a method to elucidate the host response. This study utilized a novel microarray method to interrogate the host transcriptome in feces in Malawian children with EED. Our data showed that the children studied had a range of %L values, consistent a spectrum of EED from normal to severe. We identified 12 transcripts associated with the severity of EED, including chemokines that stimulate T-cell proliferation, Fc fragments of multiple immunoglobulin families, interferon-induced proteins, activators of neutrophils and B-cells, and mediators that dampen cellular responses to hormones. EED associated transcripts mapped to pathways related to cell adhesion, and responses to a broad spectrum of viral, bacterial and parasitic microbes and enhanced phagocytosis. Several mucins, regulatory factors and protein kinases associated with the maintenance of the mucous layer were expressed less in children with EED than normal children. In conclusion, EED represents the focused activation of elements of the immune system and is associated with widespread intestinal barrier disruption. The differentially expressed transcripts may be explored as potential biomarkers. This study was carried out, firstly using Affymetrix Human Transcriptome Array 2.0 (HTA2.0, data have already been deposited in the GEO database: GSE74681) to profile the transcriptome of the host stool RNAs in 259 subjects, and then using droplet digital PCR (ddPCR) assays to validate the microarray data in terms of signal correlation. The ddPCR assay was done for a total of 60 genes in 25 - 236 (median 67) of the 258 RNA samples per RNA availability (1 of 259 samples [stool_080B] was not used for any of the 60 assays due to lack of availability). The data of the first 42 assays (genes) in the matrix sheets were used in Figure 4 for signal correlation analysis, and the data of the rest 18 assays (genes) were used in Table 4 for validation of 18 significant genes identified in biological pathway analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Jinsheng Yu

PROVIDER: E-GEOD-75653 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:We previously demonstrated that the osteopontin-c (OPNc) splice variant activates several aspects of the progression of prostate cancers. The goal of the present study was to develop cell line model to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles. Methods: PC-3 cells were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses; Results: Of 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, among then 16 were differentially expressed between PC-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Conclusions: Overall, the present study elucidated transcriptional changes of prostate cancer cells in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features. Cell Culture, OPN Plasmids and Transfection; As a model to examine the signaling pathways modulated by OPNc overexpression in prostate carcinoma, we used PC-3 cell line, which were provided by ATCC. The open reading frame of OPNc was cloned into the pCR3.1 mammalian expression vector as previously described [Tilli et al., 2012]. Transfections were performed using LipofectamineTM 2000 (Invitrogen, CA). Our previous data demonstrated that PC-3 stably transfected cells contain higher levels of protein and the mRNA OPNc splicing isoform in relation to their endogenous levels in empty vector-transfected cells. PC-3 transfected with empty vector (EV) were used as a negative control in these assays. Human Cancer Pathway Finder PCR Array The Human Cancer Pathway Finder SuperArray (PAHS-033A; Qiagen) was used to determine changes in the specific genes encoding proteins related to the main hallmarks of cancer in response to OPNc overexpression. The assay design criteria ensure that each qPCR reaction will generate single, gene-specific amplicons and prevent the co-amplification of non-specific products. The qPCR Assays used in PCR Arrays are optimized to work under standard conditions enabling a large number of genes to be assayed simultaneously. Their specificity is guaranteed by SABiosciences when RT2 SYBR Green qPCR Master Mixes are used as part of the complete PCR Array System protocol. Simultaneous gene expression analyses require similar qPCR efficiencies for accurate comparison among genes. RT2 qPCR Primer Assays are designed with an amplicon size ranging from 100 to 250 bp and with PCR efficiencies uniformly greater than 90%. Overall, more than 10 thermodynamic criteria are included in the design of each RT2 qPCR Primer Assay to ensure the most reliable and accurate results for pathway-based gene expression analysis in the PCR Array System. We analyzed mRNA levels of 84 genes related to cell cycle control, apoptosis and cell senescence, signal transduction molecules and transcription factors, adhesion, angiogenesis, invasion and metastasis; and also 5 housekeeping genes and genomic DNA contamination controls. The PCR plates were run using the CFX96 Real-Time System cycler (BioRad, Hercules, CA), following a superarray two-step cycling PCR protocol, in which each plate ran one cycle for 10 minutes at 95M-BM-0C, as well as 40 cycles of 95M-BM-0C for 15 seconds and 60M-BM-0C for 1 minute. After the superarray protocol was run for each plate, RT-PCR data analysis was performed using the website:http://www.SABiosciences.com/pcrarraydataanalysis.php, in order to compare gene expression of OPNc-overexpressing cells and empty vector transfected cells. Total RNA quality control, cDNA synthesis and the quantitative real-time RT-PCR (qRT-PCR) array were performed as recommended by the manufacturer (Qiagen). Data analysis of gene expression was performed using Excel-based PCR Array Data Analysis Software provided by manufacturer (Qiagen). Fold-changes in gene expression were calculated using the M-NM-^TM-NM-^TCT method, and five stably expressed housekeeping genes (M-NM-22 microglobulin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a, GAPDH and M-NM-2-actin) were used to normalize the level of expression. The statistical analysis was performed to compare the gene expression values for the OPNc-overexpressing cells and those transfected with empty vector. P<0.05 was considered statistically significant. Only genes showing a 1.5-fold or greater change were considered for further analysis.

Project description:We previously demonstrated that the osteopontin-c (OPNc) splice variant activates several aspects of the progression of ovarian cancers. The goal of the present study was to develop cell line model to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles. Methods: OvCar-3 cells were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses; Results: Of 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, 34, were differentially expressed between OvCar-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Conclusions: Overall, the present study elucidated transcriptional changes of ovarian cancer cells in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features. Cell Culture, OPN Plasmids and Transfection; As a model to examine the signaling pathways modulated by OPNc overexpression in ovarian carcinoma, we used OvCar-3 cell line, which were provided by ATCC. The open reading frame of OPNc was cloned into the pCR3.1 mammalian expression vector as previously described [Tilli et al., 2012]. Transfections were performed using LipofectamineTM 2000 (Invitrogen, CA). Our previous data demonstrated that OvCar-3 stably transfected cells contain higher levels of protein and the mRNA OPNc splicing isoform in relation to their endogenous levels in empty vector-transfected cells. OvCar-3 transfected with empty vector (EV) were used as a negative control in these assays. Human Cancer Pathway Finder PCR Array The Human Cancer Pathway Finder SuperArray (PAHS-033A; Qiagen) was used to determine changes in the specific genes encoding proteins related to the main hallmarks of cancer in response to OPNc overexpression. The assay design criteria ensure that each qPCR reaction will generate single, gene-specific amplicons and prevent the co-amplification of non-specific products. The qPCR Assays used in PCR Arrays are optimized to work under standard conditions enabling a large number of genes to be assayed simultaneously. Their specificity is guaranteed by SABiosciences when RT2 SYBR Green qPCR Master Mixes are used as part of the complete PCR Array System protocol. Simultaneous gene expression analyses require similar qPCR efficiencies for accurate comparison among genes. RT2 qPCR Primer Assays are designed with an amplicon size ranging from 100 to 250 bp and with PCR efficiencies uniformly greater than 90%. Overall, more than 10 thermodynamic criteria are included in the design of each RT2 qPCR Primer Assay to ensure the most reliable and accurate results for pathway-based gene expression analysis in the PCR Array System. We analyzed mRNA levels of 84 genes related to cell cycle control, apoptosis and cell senescence, signal transduction molecules and transcription factors, adhesion, angiogenesis, invasion and metastasis; and also 5 housekeeping genes and genomic DNA contamination controls. The PCR plates were run using the CFX96 Real-Time System cycler (BioRad, Hercules, CA), following a superarray two-step cycling PCR protocol, in which each plate ran one cycle for 10 minutes at 95M-BM-0C, as well as 40 cycles of 95M-BM-0C for 15 seconds and 60M-BM-0C for 1 minute. After the superarray protocol was run for each plate, RT-PCR data analysis was performed using the website:http://www.SABiosciences.com/pcrarraydataanalysis.php, in order to compare gene expression of OPNc-overexpressing cells and empty vector transfected cells. Total RNA quality control, cDNA synthesis and the quantitative real-time RT-PCR (qRT-PCR) array were performed as recommended by the manufacturer (Qiagen). Data analysis of gene expression was performed using Excel-based PCR Array Data Analysis Software provided by manufacturer (Qiagen). Fold-changes in gene expression were calculated using the M-NM-^TM-NM-^TCT method, and five stably expressed housekeeping genes (M-NM-22 microglobulin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a, GAPDH and M-NM-2-actin) were used to normalize the level of expression. The statistical analysis was performed to compare the gene expression values for the OPNc-overexpressing cells and those transfected with empty vector. P<0.05 was considered statistically significant. Only genes showing a 1.5-fold or greater change were considered for further analysis.

Project description:We investigated the roles of IRF-3 and IRF-7 in innate antiviral immunity against dengue virus (DENV). Double-deficient Irf-3-/-7-/- mice infected with the DENV2 strain S221 possessed 1,000-150,000 fold higher levels of viral RNA than wild-type and single-deficient mice 24 hours after infection; however, they remained resistant to lethal infection. IFN-α/β was induced similarly in wild-type and Irf-3-/- mice post DENV infection, whereas in the Irf-7-/- and Irf-3-/-7-/- mice, significantly low levels of IFN-α/β expression was observed within 24 hours post-infection. IFN-stimulated gene (ISG) induction was also delayed in Irf-3-/-7-/- mice relative to wild-type and single-deficient mice. In particular, Cxcl10 and Ifnα2 were rapidly induced independently of both IRF-3 and IRF-7 in the Irf-3-/-7-/- mice with DENV infection. Higher levels of serum IFN-γ, IL-6, CXCL10, IL-8, IL-12 p70, and TNF were also observed in Irf-3-/-7-/- mice 24 hours after infection, at which time point viral titers peaked and started to be cleared. Antibody-mediated blockade experiments revealed that IFN-γ, CXCL10, and CXCR3 function to restrict DENV replication in Irf-3-/-7-/- mice. Additionally, the ISGs Cxcl10, Ifit1, Ifit3, and Mx2 can be induced via an IRF-3- and IRF-7-independent pathway that does not involve IFN-γ signaling for protection against DENV. Collectively, these results demonstrate that IRF-3 and IRF-7 are redundant, albeit IRF-7 plays a more important role than IRF-3 in inducing the initial IFN-α/β response; only the combined actions of IRF-3 and IRF-7 are necessary for efficient control of early DENV infection; and the late, IRF-3- and IRF-7-independent pathway contributes to anti-DENV immunity. To identify the antiviral genes that are controlled by IRF-3 and IRF-7 signaling during DENV infection, we examined a panel of ISG expression in the spleens of wild-type, Irf-3-/-, Irf-7-/- and Irf-3-/-7-/- mice at 12 or 24 hours after DENV infection using a quantitative PCR array kit. The fold changes in expression of 55 genes from infected mice were normalized to that of strain-matched naïve mice.

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Environmental enteric dysfunction includes a broad spectrum of inflammatory responses and epithelial repair processes (RT-PCR)

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