miRNAs are differentially regulated by mTORC1 and mTORC2, and miR-9-3p is a proapoptotic miRNA induced by pp242 in multiple cell lines. (A) MCF-7 cells were treated with control, 200-nM PP242, or 100-nM rapamycin, and after 48 h total miRNAs were analyzed with microarray. This experiment was completed once. Differential expression patterns of miRNAs between the groups are shown using a matrix plot. (B) PP242 and rapamycin-responsive miRNAs (at least threefold changes in expression vs. control) are presented. (C) Mimics of several miRNAs were transfected into MCF-7 cells, followed by 20-µM cisplatin treatment or serum starvation for 24 h, and consequent cell death was monitored using trypan blue staining. (D) miR-9-3p levels of MCF-7, A549, and MDA-MB-231 cells subjected to PP242 or rapamycin treatment were assayed using RT-qPCR to verify microarray results. Phosphorylated S6 and Akt were additionally monitored using Western blotting to ensure effective and specific treatment. (E and F) MCF-7 (E) and MDA-MB-231 (F) cells were transfected with miR-9-3p mimics at different concentrations as indicated and subsequently left untreated or subjected to serum starvation or 5-FU exposure. 60 h after transfection, cells were imaged using a light microscope (left), detached with trypsin, and monitored using trypan blue staining (middle) or harvested and analyzed via Western blotting for PARP cleavage (right). Bars, 50 µm. (G and H) MCF-7 (G) and MDA-MB-231 (H) cells were transfected with miR-9-3p antagomir at various concentrations and either analyzed for PARP cleavage or death rate, as indicated. Error bars represent mean values ± SEM. C, control; ctr, control; NC, negative control.