Dataset Information

Global Proteomic Profiling in Chronic Iron-Exposed Fallopian Tube Secretory Epithelial Cells

ABSTRACT: We previously published (Oncogenesis, Rockfield et al., 2019) that chronic iron exposure (with ferric ammonium citrate, FAC) in immortalized fallopian tube secretory epithelial cells resulted in increased growth and migratory propensity. Our focused analyses identified a subset of oncogenic markers, including EVI1 (amplified at 3q26.2 in high grade serous epithelial ovarian tumors), that were altered following long term iron treatment. Herein, we have extended these studies to global proteomics analyses via a mass spectrometry (MS)-based approach.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

DISEASE(S): Malignant Neoplasm Of Ovary

SUBMITTER: Stanley Stevens

LAB HEAD: Meera Nanjundan

PROVIDER: PXD018416 | Pride | 2021-09-09

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20190403_75cmNormPep_CV_1.raw	Raw
	20190403_75cmNormPep_CV_2.raw	Raw
	20190403_75cmNormPep_CV_3.raw	Raw
	20190403_75cmNormPep_CV_4.raw	Raw
	20190403_75cmNormPep_CV_5.raw	Raw

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Publications

Global miRNA/proteomic analyses identify miRNAs at 14q32 and 3p21, which contribute to features of chronic iron-exposed fallopian tube epithelial cells.

Chhabra Ravneet R Rockfield Stephanie S Guergues Jennifer J Nadeau Owen W OW Hill Robert R Stevens Stanley M SM Nanjundan Meera M

Scientific reports 20210318 1

Malignant transformation of fallopian tube secretory epithelial cells (FTSECs) is a key contributing event to the development of high-grade serous ovarian carcinoma (HGSOC). Our recent findings implicate oncogenic transformative events in chronic iron-exposed FTSECs, including increased expression of oncogenic mediators, increased telomerase transcripts, and increased growth/migratory potential. Herein, we extend these studies by implementing an integrated transcriptomic and mass spectrometry-ba ...[more]

PMID: 33737539

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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.