RNA-Seq based transcriptome analysis of colorectal cancer cells in response to lactate treatment
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ABSTRACT: Recently, the presence of lactate in colorectal cancer (CRC) has been rediscovered as crucial signalling molecule which plays key roles in the regulation of the metabolic pathways, the immune response, and cell-to-cell communication within the tumour microenvironment. We here report the RNA-Seq based transcriptome analysis of DLD1 and SW480 cells in response to lactate treatment. By a number of statistical tests, a series of genes were statistically significant, indicating a gene set profoundly responded to lactate in CRC. To unveil the molecular mechanism associated with the effects of lactate on CRC development and progress, these genes were annotated using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses. Our data demonstrate that targeting lactate metabolism and transport represents a promising approach for CRC therapeutics.
Project description:Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) is a lipid second messenger produced by class I phosphoinositide 3-kinases (PI 3-kinases) phosphorylation of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Although typically associated with the plasma membrane, it is now appreciated that PI(3,4,5)P3 generation also occurs in the nucleus to regulate a variety of biological functions. Using a PI(3,4,5)P3-specific GST-GRP1PH domain reporter and immunofluorescence microscopy, we have found that DLD1 and SW480 colorectal cancer (CRC) cells exhibit pronounced nuclear staining of PI(3,4,5)P3. To characterise the nuclear PI(3,4,5)P3 interactome in DLD1 and SW480 cells, we performed affinity pull-down experiments on nuclear extracts using a ω-amino analogue of phosphatidylinositol 3,4,5 -triphosphate (PI(3,4,5)P3 immobilised onto Affi-gel10 beads. Gel fractionation and LC/MS-MS analysis was performed to reduce sample complexity and increase resolution, with samples run on a LTQ Orbitrap Elite mass spectrometer. DLD1 and SW480 cells were both analysed in four independent biological replicates. A protein was considered a member of the nuclear PI(3,4,5)P3 interactome if it was identified in at least two experiments (with at least two peptides, FDR of 1%) in both cell lines, yielding a total of 1237 unique proteins (representing 1103 genes for DLD1 and 1001 genes for SW480 cells).
Project description:Using genome-scale CRISPR-Cas9 screening, our study revealed KMT2A as a critical regulator of β-catenin-driven CRC progression. To determine the role of KMT2A in β-catenin-mediated transcription, control and KMT2A-ablated DLD1 and SW480 colorectal cancer (CRC) cells were subjected to CHIP-seq analysis using anti-β-catenin and anti-H3K4me3 antibodies. Data obtained from the CHIP-seq experiments indicated a key role of KMT2A in β-catenin binding on active promoters.
Project description:Dihydroartemisinin (DHA), a well-known antimalarial drug, has been widely investigated as its anti-tumor effects in multiple malignancies. However, its effects and regulatory mechanisms in colorectal cancer (CRC) are still unproved. In this study, the in vitro experiments including CCK8, EdU, Transwell, flow cytometry analyses and in vivo tumorigenesis model were conducted to assess the effects of DHA on the bio-behaviors of CRC cells. Additionally, RNA-seq combined with gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses was used to obtain the targets of DHA and these were verified by molecular docking, qRT-PCR and Western blot. As a result, we found that DHA significantly suppressed the proliferation, DNA synthesis and invasive capabilities, and induced cell apoptosis and cell cycle arrest in HCT116, DLD1 and RKO cells in vitro and in vivo. Further analyses indicated that the targets of DHA were predominantly enriched in cell cycle-associated pathways, including CDK1, CCNB1 and PLK1, and DHA could bind with CDK1/CCNB1 complex and inhibit the activation of CDK1/CCNB1/PLK1 signaling. Moreover, cucurbitacin E, a specific inhibitor of CDK1/CCNB1 axis enhanced the inhibitory effects of DHA on DNA synthesis and colony formation in HCT116 and DLD1 cells. In short, DHA could suppress the tumorigenesis and cycle progression of CRC cells by targeting CDK1/CCNB1/PLK1 signaling.
Project description:To validate the suitability of two commonly used colorectal cancer cell lines, DLD1 and SW480, as model systems to study colorectal carcinogenesis, we treated these cell lines with β-catenin siRNA and identified β-catenin target genes using DNA microarrays. The list of identified target genes was compared to previously published β-catenin target genes found in the PubMed and the GEO databases. Based on the large number of β-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T as well as the large overlap with confirmed β-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study β-catenin regulated genes and signaling pathways
Project description:To validate the suitability of two commonly used colorectal cancer cell lines, DLD1 and SW480, as model systems to study colorectal carcinogenesis, we treated these cell lines with beta-catenin siRNA and identified beta-catenin target genes using DNA microarrays. The list of identified target genes was compared to previously published beta-catenin target genes found in the PubMed and the GEO databases. Based on the large number of beta-catenin target genes found to be similarly regulated in DLD1, SW480 and LS174T as well as the large overlap with confirmed β-catenin target genes, we conclude that DLD1 and SW480 colon carcinoma cell lines are suitable model systems to study beta-catenin regulated genes and signaling pathways 12 arrays (2 cell lines, 2 treatments, 3 biological replicates)
Project description:DLD1 is an APC mut, KRAS mut, P53 mut CRC cell line. PROX1 transcription factor, target of Wnt pathway in CRC, is our protein of interest.DLD1 cells are PROX1 negative. We overexpressed through lentiviral expression PROX1 protein or the empty vector psd44, through selection of the cells in puromycin resistance. Afterwards we compared the transcriptional program of the DLD1-PROX1 and DLD1-Control cells growing in monolayer in vitro.
Project description:The objective of this study is to assess gene expression changes and identify gene sets associated with chemo-radiotherapy (CRT) in colorectal cancer (CRC) patients. We performed RNA-seq based transcriptome profiling using primary tumor samples obtained from 22 CRC patients. By a number of statistical tests, a total of 575 genes were statistically significant, indicating a gene set profoundly responded to chemo-radiotherapy in CRC. To select the genes showing strong gene expression intensities and critically validate the differences between treatment-responded and non-responded patients, we applied more rigorous cut-off for gene selection, revealing a total of 8 final gene candidates. These genes were applied to the downstream experimental assays.
Project description:L. plantarum is known to possess an L-lactate inducible lactate racemase activity (Goffin et al. 2005. J. Bacteriol. 187:6750). In the present study, microarrays were used in order to identify all genes that are up-regulated by L-lactate, but not by a racemic mixture of D- and L-lactate. A mutant of L. plantarum NCIMB8826 deficient for NAD-dependent L-lactate activity (TF101; Ferain et al. 1994. 176:596), and thus producing no L-lactate, was grown in MRS medium at 28°C until mid-exponential phase (OD600nm 0.75). The culture was then divided into 3 sub-cultures. Optically pure sodium L-lactate (200 mM) was added to the first sub-culture (TF101 + L-lac 200 mM). An equimolar mixture of sodium D- and L-lactate (100 mM each) was added to the second sub-culture (TF101 + L/D-lac 200 mM). The third sub-culture was not treated (TF101; reference sample). The three sub-cultures were further incubated at 28°C for 1h30 (a time known to be sufficient for induction of lactate racemase activity by L-lactate). Cells were harvested by centrifugation. Microarray data were used ot identify genes that are specifically induced by L-lactate (comparison of TF101 with TF101 + L-lac 200 mM), but not by DL-lactate (comparison of TF101 with T101 + L/D-lac 200 mM). There are no biological replicates.
Project description:Experiment Description RNA sequencing was performed on Candida albicans wild type cells (SC5314) grown to exponential phase on YNB Lactate, YNB Glucose, or YNB Glucose plus Lactate, and compared to exponential Candida albicans crz1 cells grown on YNB Glucose or YNB Glucose plus Lactate. Three independent experiments were performed.
Project description:L. plantarum is known to possess an L-lactate inducible lactate racemase activity (Goffin et al. 2005. J. Bacteriol. 187:6750). In the present study, microarrays were used in order to identify all genes that are up-regulated by L-lactate, but not by a racemic mixture of D- and L-lactate.