In vitro effects of resistin on epithelial to mesenchymal transition (EMT) in MCF-7 and MDA-MB-231 breast cancer cells – qRT-PCR and Westen blot analyses data

Resistin is an adipokine produced by the white adipocytes and adipose-derived macrophages, which mediates inflammation and insulin resistance Huang et al., 1997 and Renehan et al., 2008 Feb. Here, we provide data on the effect of resistin on epithelial to mesenchymal transition (EMT) in breast cancer cells in vitro. As model systems, we used human MCF-7 (low-metastatic) and MDA-MB-231 (high-metastatic) breast cancer cell lines. To optimize experimental conditions, we treated the cells with various concentrations of resistin (12.5, 25 and 50 ng/ml) for different time intervals (6 and 24 hours), and measured SOCS3 mRNA expression by using qRT-PCR analysis. Further, we used qRT-PCR and Western blot analyses to measure the expression of various epithelial (E-cadherin, claudin-1) and mesenchymal (SNAIL, SLUG, ZEB1, TWIST1, fibronectin, and vimentin) markers after resistin treatment. This data article is part of a study Avtanski et al., 2019 May, where detailed interpretation and discussion can be found.

treatment. This data article is part of a study Avtanski et al., 2019 May, where detailed interpretation and discussion can be found. © 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Effect of resistin on suppressor of cytokine signaling 3 (SOCS3) mRNA expression
Resistin is acytokine produced by the white adipose tissue (WAT) that is known to play a major role in mediating insulin sensitivity of the insulin target tissues [1,2]. Previous research by Steppan et al. [4] demonstrated that resistin treatment of 3T3-L1 adipocytes (at concentration of 20 ng/ml) markedly induced the gene expression of suppressor of cytokine signaling 3 (SOCS3). Here, we used SOCS3 as a control gene to optimize the treatment conditions of human MCF-7 and MDA-MB-231 breast cancer cells with resistin. Cells were treated with increasing concentrations of human resistin (8.3, 12.5, 25, and 50 ng/ml) for 6 and 24 hours, and resistin mRNA expression levels were measured by qRT-PCR analysis. Data were presented as fold-change of resistin treatment vs. non-treated (Control) cells at 6 and 24 hours. Results demonstrated that resistin statistically significant upregulated SOCS3 mRNA levels in both, MCF-7 and MDA-MB-231 cells, as maximal effect was reached at concentration of 12.5 ng/ml for 6 hours of treatment ( Fig. 1  Resistin induces breast cancer cells epithelial to mesenchymal transition (EMT) and stemness through both adenylyl cyclase-associated protein 1 (CAP1)-dependent and CAP1independent mechanisms. Cytokine. 2019 May; 120:155e64 [3].

Value of the data
We demonstrated that resistin induces mRNA and protein expression of mesenchymal markers in MCF-7 and MDA-MB-231 breast cancer cells, and inhibits epithelial markers expression in MCF-7 cells. We established optimal conditions for resistin treatment in human breast cancer cells in vitro as initial necessary step for performing further experiments. Suppressor of cytokine signaling 3 (SOCS3) can be used as a control gene to evaluate the responsiveness of cancer cells to resistin treatment. These data can be used to further strengthen our understanding of the role of obesity in cancer.

Effect of resistin on the expression of epithelial to mesenchymal transition (EMT) markers in breast cancer cells in vitro
Using similar treatment conditions, we evaluated the effect of resistin on the expression of various mesenchymal and epithelial markers in MCF-7 and MDA-MB-231 cells. Results from qRT-PCR analysis showed that in MDA-MB-231 cells, resistin significantly upregulated mRNA levels of key transcription factors involved in EMT (SNAIL, SLUG, ZEB1, and TWIST1) as well as their target genes, fibronectin and vimentin ( Fig. 2 and Table 2). Results from Western blot analyses using MCF-7 and MDA-MB-231 cells demonstrated that resistin significantly increased the protein expression levels of SNAIL and vimentin in both MCF-7 and MDA-MB-231 cells, as well as concomitantly suppressed the protein levels of Ecadherin and claudin-1 in MCF-7 cells ( [3] and Table 3). In order to investigate EMT transcription factor nuclear translocation events, we further performed fractioned Western blot, analyzing ZEB1 and SNAIL protein expression in separate cytoplasmic and nuclear fractions from MCF-7 and MDA-MB-231 cells treated with resistin (12.5 ng/ml for 6 hours). The results from this experiment demonstrated that resistin time-dependently induced the nuclear translocation of these two transcription factors in both, MCF-7 and MDA-MB-231 cells ( [3] and Table 4).  Table 1. Table 1 Effect of resistin on SOCS3 mRNA levels in MCF-7 and MDA-MB-231 cells. Concentration-response and time-dependent experiment using MCF-7 and MDA-MB-231 cells treated with resistin (8.3, 12.5, 25, and 50 ng/ml) for 6 and 24 hours. SOCS3 mRNA levels were measured by qRT-PCR analysis (see also Fig. 1 Corp.). The lyophilized form was reconstituted with water to a concentration of 100 mg/ml and further to a series of dilutions of 8.3, 12.5, 25, and 50 mg/ml, which were used for cell treatment.  Table 2.

Quantitative RT-PCR analyses
Total RNA was extracted by using TRIzol™ reagent (Ambion, Thermo Fisher Scientific) and chloroform, and precipitated with iso-propanol (VWR International). The precipitated RNA was washed Table 3 Effect of resistin on epithelial and mesenchymal proteins expression in MCF-7 and MDA-MB-231 cells e densitometry analysis. MCF-7 and MDA-MB-231 cells were treated with resistin (12.5 ng/ml) for 6 and 24 hours. Densitometry analyses were performed using ImageJ software (NIH). Data are presented as % change of resistin treatment compared to control (no treatment) after normalizing to GAPDH levels. p values < 0.05 are shown in bold.  Table 4 Effect of resistin on EMT-transcription factor nuclear translocation in MCF-7 and MDA-MB-231 cells e densitometry analyses. MCF-7 and MDA-MB-231 cells were subjected to resistin treatment (12.5 ng/ml) for 6 and 24 hours. Western blots were performed using separate (cytoplasmic and nuclear) protein fractions. b-Tubulin and lamin B1 were used as loading controls.

Western blot analyses
Whole cellular protein was extracted by using Pierce™ RIPA Lysis Buffer (Cat. # 89901, Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Mammalian ProteaseArrest, Cat. Cruz Biotechnology) primary antibodies and Pierce™ Goat Anti-Rabbit Horseradish Peroxidase Conjugated Secondary Antibody (Cat. # 31460, Thermo Fisher Scientific). Protein bands were visualized by using SuperSignal West Pico Chemiluminescent Substrate (Cat. # 34580, Cell Signaling Technology) and MyECL™ Imager (Thermo Fisher Scientific). Densitometry analyses were performed with ImageJ software (National Institutes of Health) and protein expression levels were presented as percentage difference compared to control (±SEM).

Statistical analyses
Quantitative RT-PCR analyses data were based on 4-6 separate experiments, as each experimental condition was set up in duplicates, and each sample was analyzed in duplicates. Densitometry analysis of the Western blot data was based on 3 different experiments. Statistical analysis were carried out using GraphPad Prism 7 software (GraphPad Software, La Jolla, CA). Data were analyzed using ANOVA. Results are expressed as mean ± SEM and are considered statistically significant if p < 0.05.