miR-181a directly targets Bim and mediates cell adhesion–induced Bim down-regulation. (A). Knockdown of miR-181a using anti–miR-181a. The cells were transfected with anti–miR-181a or a scrambled oligonucleotide control, and miR-181a was analyzed by TaqMan microRNA qRT-PCR assays. Results in fold were obtained and expressed relative to small RNA U6 expression levels in Jeko-1 cells. The mean values and SDs from 4 independent experiments are shown. The Student t test was used for statistical analysis. *P < .05. (B-C). Knockdown of miR-181a increases Bim protein (B) but not mRNA (C) level with and without HK cell adhesion. Results for panel B are representative of 3 independent experiments. Results for panel E are means ± SD of 3 independent experiments. (D) Overexpression of miR-181a in pre–miR-181a–transfected Jeko-1 cells. Jeko-1 cells were transfected with pre–miR-control or pre–miR-181a (50 nmol/L) using Amaxa's Nucleofector system, and the miR-181a expression was analyzed by TaqMan microRNA qRT-PCR assays. (E) Overexpression of miR-181a down-regulated Bim (all Bim isoforms, EL, L, and S) expression. Pre–miR-control (pre–miR-Ctrl)– or pre–miR-181a–transfected (pre–miR-181a) Jeko-1 lymphoma cells (10⁵/mL) were in suspension (Sus) or adhered to HK cells (HK-Ad) for 24 hours, and Bim expression was analyzed by Western blot. (D-E) Each are representative of at least 3 experiments with mean ± SD. Results (in fold) were obtained and expressed relative to small RNA U6 expression level. The Student t test was used for statistical analysis. *P < .05. (F) pmiR-Report.Bim 3′ UTR reporters were constructed using the miRANDA Human miRNA Targets Web site (http://cbio.mskcc.org/cgi-bin/mirnaviewer/mirnaviewer.pl) and the Targetscan (Version 5.4) Web site (http://genes.mit.edu/targetscan) based on the target prediction algorithms. Jeko-1 (G) or SUDHL-4 (H) cells were transfected with pmiR-Report control vector (Ctrl-vector) or pmiR-Report.Bim 3′ UTR wild-type (3′UTRw) or pmiR-Report.Bim 3′UTR mutant (3′UTRm) plasmids harboring point mutations in the target sites for miR-181a. These reporter plasmids were cotransfected with pre–miR-181a or anti–miR-181a (50 nmol/L) or control oligonucleotides or plasmid. The luciferase activities (in triplicate) of these transfected cells were measured 36 hours after transfection. Renilla luciferase activities were normalized against firefly luciferase activities, and mean normalized Renilla luciferase activities (± SD) from at least 3 independent experiments were determined and expressed relative to control values. In panels B and E, the relative change in Bim protein was measured by quantitative densotometry and is indicated below each lane.