Lysis further confirmed increased mTOR activity demonstrated through the increased total and phospho-mTOR (S2448) in MCF-7miR-155 cells (Figure 1D). In addition, decreased Rictor and TSC1 protein levels were observed in MCF-7-miR-155 cells (Figure 1D). The combined loss of Rictor (a critical mTORC2 component) and TSC1an mTORC2 activator and mTORC1 suppressor) suggests that miR-155 induced mTOR signaling through the mTORC1 complex. Evaluation of downstream targets of mTORC1 and mTORC2 were next evaluated. Western blot demonstrated enhanced phosphorylation of p-eEF2K and p-eIF4B downstream targets of mTORC1 (Figure 1E), there was however no noticeable change in p-p70s6K or p-S6 ribosomal protein. mTORC2 is known to enhance PKC expression, so we next evaluated PKC gene expression and saw repressed expression of PKC in the MCF-7-miR-155 cell line (Figure 1F).Stable expression of miR-155 disrupts ER signaling in MCF-7 breast Crotaline biological activity cancer cellsp value determined by size of miR-155 target data set, number of molecules in a pathway, and number of molecules in database. Determines probability that miR-155 targets are biased towards a particular pathway.As crosstalk between mTOR signaling and ER has been reported [13,14], it was of interest to investigate a possible role for miR-155 in ER signaling. We performed qPCR with an ER-responsive and breast cancer associated genes qPCR gene array. Aberrant basal expression of ER-regulated genes was observed in the MCF7-miR155 cell line compared to vector. Altered genes included PgR (a known target of ER and mTOR signaling) and PLAU (both decreased) and BCL2, ERBB2, TFF1, and SERPINA3 (all increased) (Figure 2A and Figure 2B respectively). This striking divergent expression of ER-regulated genes suggested that miR-155 acts as a possible regulator of estrogen-mediated signaling. ToMartin et al. Molecular Cancer 2014, 13:229 http://www.molecular-cancer.com/content/13/1/Page 5 ofFigure 2 Overexpression of miR-155 selectively alters estrogen stimulated gene expression of ER-regulated genes in vitro. (A-B) Gene panel array for ER regulated and cancer associated genes, n = 2. (A) Down regulated genes and (B) up regulated genes (logarithmic scale) in MCF-7-miR-155 cells compared to vector control. Results represent average fold change ?SEM. (C-F) QPCR was performed for genes, n = 5 (C) PgR, (D) SDF-1, (E) BCL2, and (F) SERPINA3. Cells were grown in 5 CS phenol free DMEM for 48 hours before treatment with DMSO or E2 (1nM) for 24 hours. Cycle number was normalized to beta actin and MCF-7-vector control cells treated with DMSO scaled to 1. Bars represent average fold change ?SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.further investigate this possibility, cells were serum starved for 48 hours, treated with 17- estradiol (E2, 1 nM) or vehicle for 24 hours, and ER target genes (PgR, SDF-1, BCL2, and SERPINA3) were analyzed by qPCR. As previously observed in our gene panel array, significantly decreased basal PgR mRNA levels were observed in MCF-7-miR-155 cells compared to MCF-7vector (Figure 2C). Basal expression of SDF-1 was also significantly lower in our MCF-7-miR-155 cell line (Figure 2D). Basal expression levels of BCL2 and SERPINA3 were significantly increased in MCF-7-miR-155 cells and E2 treatment further induced expression of both genes compared to MCF-7-vector cells (Figure 2E and Figure 2F respectively). E2 stimulation failed to increase PgR expression levels in MCF-7-miR-155 cell line to that of PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28300835 basal levels o.
