Effect of long-term education with EVs from women with obesity on MCF7 gene expression
Breast adipose tissue is an important contributor to the obesity-breast cancer link.  Dysregulated cell metabolism is now an accepted hallmark of cancer. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we found that long-term education of breast cancer cells (MCF7, T47D) with EVs obtained from breast adipose tissue of women who are overweight or obese (O-EVs) leads to sustained increased proliferative potential. RNA-Seq of O-EV-educated MCF7 cells demonstrates increased expression of genes, such as ATP synthase and NADH: ubiquinone oxidoreductase, involved in oxidative phosphorylation. Overall design: MCF7 cells were seeded at 40,000 cells/well in a 6-well plate, and treated with media containing 1mg/mL L-EVs, O-EVs or vehicle control (PBS; same volume) on day 1, 4 and 7. The culture media was refreshed with each EV treatment. On day 8, cells were harvested for further experiments. Cells were lysed using Qiazol lysis reagent (Qiagen, #79306), and total RNA was extracted from samples by Trizol lysis and RNeasy Mini Kit (Qiagen, #74106) following the manufacturer's protocols. Samples were submitted for RNA-seq at the Genomics Resources Core Facility (GRCF, Weill Cornell Medicine). Sequencing libraries were constructed at the GRCF following the Illumina TruSeq Stranded mRNA library preparation protocol (Poly-A selection and Stranded RNA-Seq). The libraries were sequenced with single read 50 bp on the Illumina NovaSeq6000 sequencing platform. All reads were independently aligned with STAR_2.4.0f1 (Dobin et al, 2013) for sequence alignment against the human genome sequence build hg19, downloaded via the UCSC genome browser and SAMTOOLS v0.1.19 (Li et al, 2009) for sorting and indexing reads. Cufflinks (2.0.2) (Trapnell et al, 2012) was used to estimate the expression values (FPKMS), and GENCODE v19 (Derrien et al, 2012) GTF file for annotation.