Fig. 4

c-Myc enhances Kaposin B to regulate microRNA promoter activity. a Knockdown of endogenous c-Myc levels in Kaposin B(+) HUVECs rescues miR-221 (left) and miR-222 (right) expression. b ChIP analysis with immunoglobulin G control or anti-FLAG antibody (detecting Kaposin B). (upper) Schematic representation of miR-221/-222 promoter region and PCR fragments. (lower) HMEC1 cells stably expressing empty vector, Kaposin B, or Kaposin B + c-Myc were subjected into ChIP assays. The −2600 promoter region PCR product was used as a negative control. c Wild type (−1600) promoter construct or double mutant construct (upper) was transfected into a HMEC1 cell line stably expressing Kaposin B (HMEC1-Kaposin B), and ChIP-qPCR assays were performed on transfectants using anti-FLAG mAb. d Endogenous c-Myc was stably knocked down using shRNA in a HMEC1-Kaposin B stable cell line, and cells with or without c-Myc knockdown were subjected to ChIP assays and qPCR for indicated regions (n = 3). e Reporter activity of tested reporter plasmids after co-transfection with Kaposin B, c-Myc, or c-Myc + Kaposin B. Schematic representation of reporter constructs (left) and reporter assay results (n = 3) (right). *: P < .05. f Reporter assays on E1 or E2/3 single mutants, and on the E1 and E2/E3 E-boxes double mutant. Only the double mutant showed significant promoter activity restoration. (n = 3) *: P < .05