Unknown,Transcriptomics,Genomics,Proteomics

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Mouse primary astrocytes: LCN2 protein treated vs. control

ABSTRACT: Transcriptional profiling of mouse primary astrocytes comparing control untreated astrocytes with astrocytes treated with recombinant LCN2 protein (10 micro gram/ml). Goal was to determine the effects of LCN2 treatment on global gene expression in astrocytes. A secreted protein lipocalin-2 (LCN2) has been implicated in diverse cellular processes including cell morphology and migration. We have previously demonstrated that lcn2 mediates reactive astrocytosis. In order to further understand the role of lcn2 in the CNS, astrocyte transcriptome was analyzed following LCN2 treatment. Chemokines were the major group of genes upregulated by LCN2. Two-condition experiment, control untreated astrocytes vs. LCN2 protein treated astrocytes. Biological replicates: 1 control replicates, 1 treated replicates.

ORGANISM(S): Mus musculus

SUBMITTER: Kyoungho Suk

PROVIDER: E-GEOD-15667 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Iron is essential for many cellular processes and is required by bacteria for replication. To acquire iron from the host, pathogenic Gram-negative bacteria secrete siderophores, including Enterobactin (Ent). However, Ent is bound by the host protein Lipocalin 2 (Lcn2), preventing bacterial reuptake of aferric or ferric Ent. In two experiments we treated A549 (lung cancer cell line) cells with Lcn2, Ent, and iron, alone and in combination. In experiment 1, biological duplicates of 4 conditions were used: PBS control, Lcn2, Lcn2+Ent, and Lcn2+Ent+iron. In experiment 2, 4 biological replicates of 4 conditions were used: PBS control, Ent, iron, and Ent+iron. Targets made from the samples were hybridized to Affymetrix Human Gene 1.0 ST arrays to measure transcript abundances. The RMA algorithm was used to estimate transcript levels. Replicate samples were exchangeable, so we fit one-way ANOVA models to log2-transformed data separately to each experiment, and tested for pairwise differences between groups in each experiment, as well as asking if the Ent vs. PBS differences were larger or smaller than the Ent+iron vs. iron differences (Ent by iron interactions). We report results for 29096 probe-sets that were not annotated as positive or negative controls on the array. A supplementary Excel workbook is provided that contains the estimated expression level, some probe-set annotation, and simple statistical analysis for each probe-set. It may be convenient for some users, however obtaining newer probe-set annotation may be advisable. A549 (ATCC CCL-185) cells, a human type II pneumocyte cell line, were cultured in F12K (Invitrogen, Carlsbad, CA) media supplemented with 10% fetal bovine serum (Invitrogen) and 1:100 penicillin streptomycin (Invitrogen). 24-well plates were seeded with A549 cells at a concentration of 35000 cells/well. After two days, cells were weaned from serum and antibiotics overnight. Cells were then stimulated overnight with the indicated combinations of 50 uM Ferric ammonium citrate (FAC) (Sigma, St. Louis, MO), 50 um Ent (Sigma or EMC Microcollections, Tubingen, Germany), and/or 25 uM lipocalin 2 (Lcn2) in F12K media lacking serum or antibiotics. Prior to incubation with cells, siderophore-Lcn2 complexes were prepared by sequential incubation at room temperature of FAC and Ent for 30 minutes followed by addition of Lcn2 and incubation for an additional 30 minutes. In experiment 1, 2 biological replicates of each of these 4 conditions were grown: PBS, 25 uM lipocalin 2 (Lcn2), a combination of 25 uM Lcn2 with 50 uM Enterobactin (Ent), and a combination of Lcn2, Ent and 50 uM Ferric Ammonium Citrate (FAC). In experiment 2, 4 biological replicates of each of these 4 conditions were grown: PBS, 50 uM Ent, 50 uM FAC, and a combination of 50 uM Ent and 50 uM FAC. Cells were harvested and RNA was extracted using an miRNeasy Mini Kit (Qiagen). Biotinylated cDNA targets were prepared according to the Ambion WT expression kit protocol from 150 ng total RNA. There are no batches within each experiment - the samples in each group are exchangeable. Targets were hybridized to Affymetrix Human Gene 1.0 ST arrays. Arrays were scanned and quantified by usual procedures, and transcript abundances for 29096 non-control probe-sets estimated by an RMA algorithm.

Project description:Background: Distant metastasis remains a major reason for the high recurrence and mortality of colorectal cancer (CRC). However, the underlying molecular mechanisms driving metastasis in CRC remain poorly understood. In this study, we investigated the mechanisms underlying the inhibitory effects of lipocalin-2 (LCN2) on CRC metastasis. Methods: We assessed the expression and clinical significance of LCN2 in human CRC specimens and CRC cell lines using, immunohistochemistry, and western blot analyses. We evaluated the migratory and invasive capabilities of CRC cells influenced by LCN2 using in vitro transwell assays and in vivo lung metastatic models. RNA sequencing and proteome analysis were employed to identify potential downstream targets of LCN2. Rescue experiments were conducted to further elucidate the potential mechanisms of LCN2 and its downstream effectors in CRC. Results: LCN2 exhibited high expression levels in human CRC tissues and an inverse correlation with N classification, advanced AJCC stages, and shorter overall survival. LCN2 expression independently predicted a more favorable outcome for CRC patients. Upregulation of LCN2 effectively suppressed CRC cell metastasis both in vitro and in vivo. Mechanistically, Transforming growth factor beta 1 (TGFB1) and C-X-C motif chemokine ligand 5 (CXCL5) were identified as downstream effectors of LCN2, with LCN2 inhibiting CRC metastasis through repression of the TGFB1/CXCL5 axis. Furthermore, either TGF-βR1 inhibitor SB431542 or CXCR2 antagonist SB225002 treatment moderately decreased the migratory and invasive capabilities of DLD-1-LV-shLCN2 cells, whereas the combination treatment of the two agents dramatically decreased the migratory and invasive capabilities of DLD-1-LV-shLCN2 cells. Conclusions: This study underscores LCN2 as an independent protective factor and prognostic biomarker for CRC patients. Combined treatment with the SB431542 and the SB225002 significantly attenuated LCN2-related CRC metastasis. Targeting the LCN2/TGFB1/CXCL5 axis emerges as a promising therapeutic strategy for managing LCN2-related metastatic CRC.

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Mouse primary astrocytes: LCN2 protein treated vs. control

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