miR-124a alters cancer-related genes while specifically targeting STAT3. (A) Heat map (left) and fold-expression (right) of genes altered by miR-124a–induced 72 hours. Fold change is calculated compared with pTRIPZ–induced ED-40515(–) cells. Bold, dotted lines mark a onefold loss or gain in fold expression. Genes with “*” are known to be altered by STAT3. (B) Illustration of the miR-124a–binding sites in the 3′-UTR of STAT3 (top). In silico analysis of the top 20 altered genes in ED-40515(–) TET-On miR-124a cells (shaded areas are possible STAT3 binding sites). (C-D). pCDNA, miR-124a/pCDNA, or mutant miR-124a/pCDNA (C-mutated miR-124a sequence) were transfected into 293T cells along with wild-type or mutant (D) STAT3-UTR-pGL3 and the RL-TK plasmid. Forty-eight hours after transfection, cell lysates were measured for firefly (STAT3 3′UTR) and renilla (RL-TK, internal control) activity. All luciferase assays was performed at least twice. (E) Detection of STAT3 in stable 293T-pTRIPZ or –miR-124a cells induced 72 hours with 2 μg/mL Dox. (F) 293T-pCDNA and –wild-type-STAT3 cells were established under puromycin selection. Cells were then transfected with either 50 nM miR-124a oligo or a control oligo (Negt#1). One week after transfection, cells were stained for cell growth (left). Results represent 1 of 2 experiments performed. The overexpression of STAT3 was confirmed (right). (G-H) Stable miR-124a lines were established in MT4, MT1, Tl-Om1, ED-40515(–), and Nalm-20, Molt4, Tanoue cell lines. miR-124a expression was confirmed after 72 hours’ induction with 2 μg/mL Dox (G), along with loss of STAT3 (H).