IGF-1 Represses miRNA Expression.

<p>MCF-7 cells were grown in 5% charcoal stripped DMEM for 48 hours. Cells were treated for 2, 5, or 24 hours with 50 ng/ml IGF-1 or vehicle. Cells were collected for total RNA extraction and q PCR was performed for (A) total miR-let-7c, let-7g, miR-15b, miR-98, and miR-195 and (B) pre-let-7c, pre-let-7g, pre-mir-15b, pre-mir-98, pre-mir-195. Normalization was to U6 and vehicle treated cells designated as 1. Error bars represent SEM, n≤5. * Significantly different from IGF, p≤0.05.</p

Insulin-Like Growth Factor-1 Signaling Regulates miRNA Expression in MCF-7 Breast Cancer Cell Line

<div><p>In breast carcinomas, increased levels of insulin-like growth factor 1 (IGF-1) can act as a mitogen to augment tumorigenesis through the regulation of MAPK and AKT signaling pathways. Signaling through these two pathways allows IGF-1 to employ mechanisms that favor proliferation and cellular survival. Here we demonstrate a subset of previously described tumor suppressor and oncogenic microRNAs (miRNAs) that are under the direct regulation of IGF-1 signaling. Additionally, we show that the selective inhibition of either the MAPK or AKT pathways prior to IGF-1 stimulation prevents the expression of previously described tumor suppressor miRNAs that are family and cluster specific. Here we have defined, for the first time, specific miRNAs under the direct regulation of IGF-1 signaling in the estrogen receptor positive MCF-7 breast cancer cell line and demonstrate kinase signaling as a modulator of expression for a small subset of microRNAs. Taken together, these data give new insights into mechanisms governing IGF-1 signaling in breast cancer.</p> </div

miRNA Expression Altered by IGF-1 Treatment in MCF-7 Breast Cancer Cells.

<p>Results represent fold change. “-” represents no significant change between vehicle treated MCF-7 cells and drug treatment.</p

miRNA families regulated by MAKP or PI3K/AKT signaling.

<p>miRNA families regulated by MAKP or PI3K/AKT signaling.</p

IGF-1 regulates miRNAs through MAPK and PI3K/AKT signaling pathways.

<p>(A) MCF-7 cells were grown in 5% charcoal stripped DMEM for 48 hours. Cells were treated with UO1261 for 30 minutes prior to 24 hours of treatment withvehicle. Q RT- PCR was performed for miRNAs miR-15b, miR-98, let-7c, let-7g, and miR-195. Normalization was to U6 and vehicle treated cells designated as 1. (B) MCF-7 cells were grown in 5% charcoal stripped DMEM for 48 hours. Cells were treated with LY294002 for 30 minutes prior to IGF treatment for 24 hours or vehicle. Normalization was to vehicle treated cells and error bars represent SEM, n≤5. * Significantly different from vehicle treated cells p≤0.05.</p

IGF-1 induced miRNA expression in MCF-7.

<p>Microarray analysis was performed on MCF-7 cells. Cells were grown in 5% charcoal stripped DMEM for 48 hours. Cells were then treated with 50 ng/mL IGF-1, 5 µM LY294002 or 10 µM PD98059 for 18 hours. Cells with combined IGF-1/LY294002 or IGF-1/PD98059 were pre-treated with inhibitor for 30 minutes followed by 18 hours of stimulation with IGF-1. Results represent quadruplicate internal repeats.</p

The effect of BPA and DDT on endogenous miR-21 target genes in MCF-7 cells.

<p>MCF-7 cells were treated for 18 hrs. with 0, 1, 10 and 25 µM BPA or DDT followed by RT-PCR of PDCD4 and maspin.</p

MicroRNA microarray heatmaps of MCF-7 cells treated with vehicle, estrogen, BPA, or DDT.

<p>(A) Estrogen versus control (B) BPA versus control and (C) DDT versus control. Arrowheads represent expression of miR-21.</p

Effects of E₂, BPA and DDT on miR-21 gene expression in MCF-7 and MCF-7F cells.

<p>MCF-7 cells (A) and MCF-7F cells (B) were treated with either vehicle, 1 nM E₂, 10 µM BPA, or 10 µM DDT. Total microRNA was extracted followed by RT-PCR. * = p<0.05, ** = p<0.001.</p

Quantitative PCR confirmation of select genes altered by estrogen, BPA, and DDT in MCF-7 cells.

<p>MCF-7 cells were treated with drug as indicated and total RNA was isolated, reverse-transcribed into cDNA, and subjected to real-time RT-PCR analysis for quantitation. Results are shown for (A) SERPINB5 (maspin) and (B) Bcl2 gene expression. Treatment of MCF-7 cells was as follows: Ethanol (vehicle), 1 nM E₂, 10 µM BPA, and 10 µM DDT. Results are expressed as the mean fold induction ± S.E.M. (***, <i>p</i><0.001; **, <i>p</i> = 0.01 – 0.001; *, <i>p</i> = 0.05 - 0.01).</p