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Novel dynamic network analysis approach characterizes progressive and complex effects of weight gain and obesity on breast cancer signaling

ABSTRACT: ER-positive tumor data was derived by RNA-sequencing of formalin-fixed paraffin-embedded specimens (FFPE) collected from 44 women diagnosed with ER+ and progesterone receptor-positive but HER2 negative (ER+/PR+/HER2-) breast cancer at the Karmanos Cancer Institute. According to a pathologist review, all tumor specimens were invasive cancer with 70% or greater cancer cell content. Patients had not received neoadjuvant cancer therapy, nor were they treated for diabetes with systemic drugs (e.g., metformin) before surgical resec- tion. Following manufacturer instructions, the Recoverall Nucleic Acid Isolation Kit for FFPE Tissue (Ambion, Austin, TX) was used to isolate RNA. Quantitation and fragmentation analysis were performed using the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and Bioanalyzer 2100 and RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA). A Qubit fluorometric assay (Thermo Fisher Scientific) was used to quantitate and equalize dsDNA prepared using the Truseq RNA Access library preparation kit (Illu- mina, San Diego, CA). Libraries were randomized, indexed, multiplexed, and paired end sequenced (200 cycles total) on an Illumina Nextseq500. Bioinformatic quality control was done using the FastQC software (v0.11.4), adapter and low-quality reads were removed using Trimmomatic v0.36. FPKM normalization and aligning were done using TopHat v2.1.1.

ORGANISM(S): Homo sapiens

PROVIDER: GSE189757 | GEO | 2022/12/31

REPOSITORIES: GEO

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Project description:Purpose: Zinc Finger MIZ-Type Containing 1 (Zmiz1) is a member of the PIAS family of protein and function as a transcriptional coactivator of Notch, Androgen Receptor (AR), p53, Estrogen Receptor (ER), and Smad3/4 . Despite Zmiz1 critical role in angiogenesis, its role in lymphatic vasculature is unknown. Here, we use HDLECs cell line to profile transcriptional changes upon Zmiz1 knockdown using siRNA. Methods: Total RNA was extracted from control and Zmiz1 siRNA transfected HDLECs using GeneJET RNA Purification Kit (Thermo Fisher Scientific, K0732) following the manufacturer instructions. RNA concentration and RNA integrity (RIN) number were determined using Qubit RNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32852) and Bioanalyzer RNA 6000 Nano assay kit (Agilent, 5067-1511) respectively. RNA library was prepared using TruSeq RNA Library Prep Kit v2 (Illumina, RS-122-2001) according to the manufacturer instructions. mRNA library was quantified using Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific, Q32851) and verified using the Bioanalyzer DNA1000 assay kit (Agilent, 5067-1505). Verified samples were sequenced using the NextSeq1000/2000 P2 Reagents (200 Cycles) v3 (Illumina, 20046812) on a Nextseq1000/2000. Sequenced reads were aligned to the human (hg19) reference genome with RNA-Seq alignment tool (STAR aligner). The aligned reads were used to quantify mRNA expression and determine differentially expressed genes using the RNA-Seq Differential Expression tool (version 1.0.1). Both alignment and differential expression analysis were performed using the tools in the illumina BaseSpace Sequence Hub. Results: We found 2,228 differentially expressed genes of which 1,115 genes were upregulated while 1,113 genes were downregulated. Downregulated genes were enriched in biological processes such as lymph vessel development, cell migration and heart valve development. Conclusions: We identify Zmiz1 regulates Prox1 in lymphatic endothelial cells

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Novel dynamic network analysis approach characterizes progressive and complex effects of weight gain and obesity on breast cancer signaling

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