The Role of miR-206 in the Epidermal Growth Factor (EGF) Induced Repression of Estrogen Receptor- (ER) Signaling and a Luminal Phenotype in MCF-7 Breast Cancer Cells

Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Table S1 - Primer Sequences Used For Real-Time PCR Detection of mRNA Transcript Levels.
  This table lists the primer sequences used in the quantitative real-time PCR detection of endogenous mRNA levels of a particular gene of interest in the MCF-7 and MDA-MB-231 breast cancer cell lines. All primers pairs generated, using both Primer3 on-line software and the NCBI database of annotated gene sequences, were optimized to have a Tm of 55 ^oC or higher, a GC content between 50 – 55%, and to have at least one annealing region spanning an exon-exon junction of the corresponding transcript.
• Supplemental Table S2 - Sequences of Inserts Used in the Various miR-206 Luciferase Reporter Constructs.
  The table above lists the oligonucleotides used to create the miR-206 firefly luciferase reporter constructs. These sequences (shown as paired duplexes) are the endogenous SRC-1, SRC-3, and GATA-3 regions that encompass the miR-206 putative target sites mentioned earlier. Flanked SacI/NheI restriction sites are depicted in bold, underlined text. The light gray region highlights the putative miR-206 binding site, while mutations within this region are illustrated as bold text.
• Supplemental Figure S1 - EGF Represses GATA-3 mRNA Levels in Both a Dose and Time-Dependent Manner.
  Hormone-depleted MCF-7 cells were treated with 0-200 ng/ml EGF for 48 h (A), or 200 ng/ml EGF for 0-32 h (B), and total RNA was harvested for subsequent real-time PCR analysis of GATA-3 and ERβ levels. Values generated from these real-time PCR experiments were normalized to RPL-19 and reported as the mean of three independent experiments ± SEM, which was set relative to the hormone-depleted or 0 h condition. (*) indicates P < 0.05, as compared to the respective controls from each experiment.
• Supplemental Figure S2 - EGF Abrogates the 17β-Estradiol-Mediated Repression of miR-206 in MCF-7 Cells.
  (A) Real-time quantitative PCR was performed to detect miR-206 levels in hormone-depleted MCF-7 cells treated with either 1 nM 17β-estradiol, 10 nM PPT, 10 nM DPN, 1 nM progesterone, and/or 100 ng/ml EGF for 48 h. Values were normalized to 5S, set as relative to the hormone-depleted treatment, and reported as the mean ± SEM of three independent experiments with four replicates per experiment, where (*) represents a P value of < 0.05. (B) To determine that MCF-7 cells expressed EGFR, Western Blot analysis was performed on duplicate samples from three individual MCF-7 and MDA-MB-231 protein lysates (left panel). The blots depicted here are representative images, while numbers below the bands represent the average densitometry readings of six replicates. β-actin served as a loading control and values were set as relative to the MDA-MB-231 protein lysates. Quantitative real-time PCR was performed to confirm the low, yet detectable, expression of EGFR in MCF-7 cells (right panel). Values were normalized to RPL-19 and are reported as the mean ± SEM of three independent experiments with four replicates per experiment, and set as relative to EGFR expression in MDA-MB-231 cell lysates.