Characterization of the molecular mechanisms underlying increased mucus production of HT29-MTX-E12 cells grown on a semi-wet interface with mechanical stimulation
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PROVIDER: PRJNA726886 | ENA |
REPOSITORIES: ENA
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Project description:The multifunctional intestinal mucus layer plays a crucial role in human health. Our understanding of the human colonic mucus layer in terms of structure, function and has been largely dependent on expensive and advanced ex vivo or in vitro models, which often require high expertise. The mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used in more simple in vitro models, but produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured in Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2 and had a thicker mucus layer compared to conventional culturing methods. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased mucus production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell inserts for 15 days were subject to transcriptome analysis to investigate underlying molecular pathways at gene expression level. We also further characterized the model by measuring transepithelial resistance and pH and lactate production of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms in which mucus production is enhanced under SWMS and propose potential applications of this model in future studies.
2021-12-06 | GSE173729 | GEO
Project description:The HT29 derivative cell line HT29-MTX-E12 (E12) produces an adherent mucus layer predominantly of the gastric MUC5AC mucin when grown on transwells. This mucus layer supports Helicobacter pylori survival in culture. E12 cells were infected with H. pylori and the transcriptome of infected and uninfected E12 were compared. Also included for comparison was the HT29 parent cell line grown on transwells. The study focused on glycosylation related genes as these regulate the structures that have a major role to play in pathogen-host cell interaction.
2017-09-21 | GSE74492 | GEO
Project description:The HT29 derivative cell line HT29-MTX-E12 (E12) produces an adherent mucus layer predominantly of the gastric MUC5AC mucin when grown on transwells. E12 cells were infected with C. jejuni and the transcriptome of infected and uninfected E12 were compared. The study focused on glycosylation related genes as these regulate the structures that have a major role to play in pathogen-host cell interaction. This experiment is related to another experiment where the cell line (E12) is replaced by the parental cell line (HT29) and importantly HT29 cells do not produce an adherent mucus layer.
2019-12-31 | GSE107565 | GEO
Project description:The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.
| S-EPMC8687553 | biostudies-literature
Project description:Glycosylation-related gene expression in the mucous-secreting gastrointestinal cell line HT29-MTX-E12 in response to infection by Campylobacter jejuni
| PRJNA420648 | ENA
Project description:HT29 cells were infected with C. jejuni 11168 and the transcriptome of infected and uninfected HT29 were compared by micoarray analysis (Affymetrix). The study focused on glycosylation related genes as these regulate the structures that have a major role to play in pathogen-host cell interaction. This experiment is related to another experiment where the parental cell line (HT29) is replaced by the derivative cell line (E12). E12 cells secrete an adherent mucus layer whereas HT29 cells do not.
2019-12-31 | GSE107563 | GEO
Project description:AimTo identify glycosylation-related genes in the HT29 derivative cell line, HT29-MTX-E12, showing differential expression on infection with Helicobacter pylori (H. pylori).MethodsPolarised HT29-MTX-E12 cells were infected for 24 h with H. pylori strain 26695. After infection RNA was isolated from both infected and non-infected host cells. Sufficient infections were carried out to provide triplicate samples for microarray analysis and for qRT-PCR analysis. RNA was isolated and hybridised to Affymetrix arrays. Analysis of microarray data identified genes significantly differentially expressed upon infection. Genes were grouped into gene ontology functional categories. Selected genes associated with host glycan structure (glycosyltransferases, hydrolases, lectins, mucins) were validated by real-time qRT-PCR analysis.ResultsInfection of host cells was confirmed by the isolation of live bacteria after 24 h incubation and by PCR amplification of bacteria-specific genes from the host cell RNA. H. pylori do not survive incubation under the adopted culture conditions unless they associate with the adherent mucus layer of the host cell. Microarray analysis identified a total of 276 genes that were significantly differentially expressed (P < 0.05) upon H. pylori infection and where the fold change in expression was greater than 2. Six of these genes are involved in glycosylation-related processes. Real-time qRT-PCR demonstrated significant downregulation (1.8-fold, P < 0.05) of the mucin MUC20. REG4 was heavily expressed and significantly downregulated (3.1-fold, P < 0.05) upon infection. Gene ontology analysis was consistent with previous studies on H. pylori infection.ConclusionGene expression data suggest that infection with H. pylori causes a decrease in glycan synthesis, resulting in shorter and simpler glycan structures.
| S-EPMC5645615 | biostudies-literature
Project description:To identify key biological pathways that define toxicity or biocompatibility after nanoparticle exposure, three human cell types were exposed in vitro to two high aspect ratio nanoparticles for 1 hr or 24 hr and collected for global transcriptomics. Transcriptional responses were measured by global microarray analysis of cells in culture. Groups (N=3 biological replicates) of Caco-2/HT29-MTX cells exposed to 0, 10 or 100 ug/ml MWCNT or TiO2-NB nanoparticles for 1 or 24 hr.
2012-12-15 | E-GEOD-42066 | biostudies-arrayexpress
Project description:The abstract of the associated publication (Selga E, Noé V, Ciudad CJ. Biochemical Pharmacology 2007 Sep 8; Article in press) is the following:; While studying differentially expressed genes between sensitive and 10-5 M Methotrexate (MTX) resistant HT29 human colon cancer cells, we identified some members of the aldo-keto reductase (AKR) superfamily. The study was followed with the member AKR1C1 (EC 1.1.1.213), validating its increase in mRNA and protein levels in MTX resistant cells. The genomic content for AKR1C1 remained unchanged between sensitive and resistant cells, thereby excluding a mechanism of AKR1C1 gene amplification. Thus, we cloned the AKR1C1 human promoter and performed luciferase experiments that revealed a transcriptional regulation of the gene in the resistant cells. Computational studies showed a putative binding site for the transcription factor Sp1. The co-transfection of Sp1 or Sp3 with different constructs of AKR1C1 promoter deletions, including and excluding the proximal GC-box, demonstrated a key role for these factors in regulating AKR1C1 transcriptional activity. Gel-shift assays revealed an increase in Sp1 and Sp3 binding in resistant compared to sensitive cells, without differences in Sp1 protein levels. Dephosphorylation of the extracts coincided with a decrease in Sp1 binding, which is consistent with a process of regulation of Sp1 by phosphorylation. We also investigated the possible relationship between AKR1C1 expression and MTX action. Overexpression of AKR1C1 counteracted the S-phase accumulation of cells and apoptosis caused by MTX treatment. This suggests a role of AKR1C1 in cell proliferation. Finally, overexpression of AKR1C1 in MTX sensitive HT29 cells conferred resistance to the chemotherapeutic agent and silencing of AKR1C1 by means of iRNA technology sensitized the cells to MTX. Experiment Overall Design: Two cell lines are compared in the study, which are HT29 colon cancer cells sensitive to methotrexate and HT29 cells resistant to 10e-5M methotrexate. Six samples are provided, which correspond to triplicates of each cell line. The samples provided were subsequently normalyzed and analyzed using the specific software GeneSpring GX 7.3.1
2008-06-16 | E-GEOD-9412 | biostudies-arrayexpress
Project description:A summary of the work associated to these microarrays is the following: Methotrexate (MTX) is one of the earliest cytotoxic drugs used in cancer therapy, and despite the isolation of multiple other folate antagonists, methotrexate maintains its significant role as a treatment for different types of cancer and other disorders. The usefulness of treatment with methotrexate is limited by the development of drug resistance, which may be acquired through different ways. To get insights into the mechanisms associated with drug resistance and sensitization we have performed a functional analysis of genes deregulated in methotrexate resistant cells, either due to its co-amplification with the DHFR gene or as a result of a transcriptome screening using microarrays. Genes adjacent to dhfr locus and included in the 5q14 amplicon were overexpressed in HT29 MTX-resistant cells. Treatment with siRNAs against those genes caused a slight reduction in cell viability in both HT29 sensitive and resistant cells. On the other hand, microarray analysis of HT29 and HT29 MTX resistant cells unveiled overexpression of caveolin 1, enolase 2 and PKCa genes in treated cells without concomitant copy number gain. siRNAs against these three genes effectively reduced cell viability and caused a decreased MTX resistance capacity. Moreover, overexpression of E-cadherin, which was found underexpressed in MTX-resistant cells, also sensitized the cells toward the chemotherapeutic agent. We provide functional evidences indicating that caveolin 1 and E-cadherin may play a critical role in cell survival and may constitute potential targets for coadjuvant therapy. Keywords: DHFR, Methotrexate, drug resistance
2008-09-08 | GSE11440 | GEO