(A) miR-548m expression inversely correlated with c-Myc expression in tetracycline-treated (Myc off) and untreated (Myc on) P493-6 cells after addition and withdraw of tetracycline. (B) Inhibition of c-Myc and EZH2 induced miR-548m expression in HBL-2 and SUDHL-4 cells. (C) Schematic diagram of the location of Myc-binding sites of the miR-548m regulatory region. S1 and S2 represent Myc-binding sites with E-box sequence. The black bar represents the miR-548m locus. NC, negative control. Both S1 and S2 are highly conserved in their putative promoter region. ChIP assay shows c-Myc and EZH2 enrichment on miR-548m promoter in c-Myc on and c-Myc off P493-6 cells and Jeko-1 cells. (D) Ectopic expression of miR-548m downregulated c-Myc expression (left) and directly targeted 3′-UTR of c-Myc (right). Jeko-1 or SUDHL-4 cells were transfected with pmiR-Report control vector (Ctrl) or pmiR-Report.Myc 3′-UTR wild-type or pmiR-Report.Myc 3′-UTR mutant (Mut) plasmids harboring point mutations in the target sites for miR-548m. The luciferase activities were normalized against firefly luciferase activities. Data are representative of at least 3 independent experiments (mean ± SD). (E) Cell adhesion to HK cells induced upregulation of c-Myc and EZH2. (F) Silencing of c-Myc with JQ1 (1 μM, 48 hours) inhibited c-Myc and stroma-induced c-Myc upregulation and increased miR-548m expression. (G) Overexpression of miR-548m with pre–miR-548m decreased stroma-induced c-Myc expression, and knockdown of miR-548m by anti–miR-548m increased c-Myc expression. Results are mean ± SD or representative from at least 3 biological replicates.