Induction of apoptosis in a dose-dependent manner in MCF-7 cells which
Induction of apoptosis in a dose-dependent manner in MCF-7 cells which was attenuated by transfection with pre-miR-10b (Fig. 3a). In MCF7TR cells, while non-specific anti-miRNA transfected cells did not exhibit much induction of apoptosis, transfection of anti-miR-10b resulted in a dose-dependent induction of apoptosis (Fig. 3b), again suggestive of sensitization of these cells to tamoxifen through deregulation of miR-10b. We also looked at the effect of miR-10b expression on invasive potential. Pre-miR-10b transfected MCF-7 cells were significantly much more invasive (Fig. 3c) while anti-miR-10b transfected MCF7TR cells were significantly less invasive, compared to respective controls (Fig. 3d). Collectively, these results provided a clear functional involvement of miR-10b in tamoxifen resistance.HDAC4 is a novel target of miR-10bHaving established a role of miR-10b in tamoxifen resistance, we next studied the molecular mechanism of such action of miR-10b by looking at its potential targets. We started with an Ingenuity Pathway Analysis to list the potential targets of miR-10b. A number of targets such as CD44, TWIST, HOXA1, HOXD10, HDAC4, PKD1, KLF4, etc. were found (Fig. 4a). We further scanned TargetScan/microRNA.org as well as reported literature for the potential targets of miR-10b and tested whether the potential targets were differentially expressed in MCR-7 vs. MCF7TR cells. Based on such screening, we focused on HDAC4, and the results presented in Fig. 4b show an alignment of miR-10b with PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26795252 its predicted site on HDAC4s 3 UTR. Next, we performed luciferase assays to confirm binding of miR-10b to 3UTR of HDAC4. MCF-7 cells were co-transfected with pre-miR-10b (or control pre-miRNA) and pEZX-MT05 vector that carried the cloned HDAC4 3UTR sequence. As can be seen in Fig. 4c, the luciferase activity was inhibited in cells transfected with pre-miR-10b by almost 50 , compared to the control cells. These results suggested a direct binding of miR-10b to 3UTR of HDAC4. ConsistentFig. 2 Effect of miR-10b levels on response to tamoxifen. a Ectopic over-expression of miR-10b in MCF-7 and T47D cells, through transfections with pre-miR-10b oligonucleotides, increased tamoxifen resistance, (b) FPS-ZM1 dose silencing of miR-10b in MCF7TR cells, through transfections with anti-miR-10b oligonucleotides, decreased their tamoxifen resistance and (c) ectopic over-expression of miR-10b in MCF-7 and T47D cells significantly attenuated tamoxifen-induced inhibition of migration potential. Cells were treated with indicated doses of tamoxifen for 48 h. *p < 0.05, **p < 0.Ahmad et al. BMC Cancer (2015) 15:Page 5 ofFig. 3 Effect of miR-10b levels on apoptosis-induction and invasion. Effect of miR-10b levels on apoptosis-induction in (a) MCF-7 and (b) MCF7TR cells. Induction of apoptosis was assessed by DNA Histone-ELISA assay. Invasion of (c) MCF-7 and (d) MCF7TR cells was assessed by plating cells in matrigel-coated plates MCF-7, non-specific pre-miRNAs transfected MCF-7 cells; MCF-7 + pre-miR-10b, pre-miR-10b transfected MCF-7 cells; MCF7TR, non-specific anti-miRNAs transfected MCF7TR cells; MCF7TR + anti-miR-10b, anti-miR-10b transfected MCF7TR cells.Fig. 4 HDAC4 is a target of miR-10b. a Ingenuity Pathway Analysis for targets of miR-10b. HDAC4 is shown with an arrow. b Sequence complementarities of miR-10b and its target HDAC4. c Luciferase assay was conducted to confirm that HDAC4 is a direct target of miR-10b. MCF-7 cells were co-transfected with dual luciferase.
