Identification of novel miR-155 target mRNAs in WM. (A) BCWM1 or MEC1 cells were treated with 20μM anti–miR-155 or scramble control for 48 hours. miR-155 inhibition decreases BCWM1 and MEC1 cell proliferation in vitro as evaluated by the [H]³ thymidine uptake assay. Experiments were performed in triplicate and repeated 3 times. Data are shown as means ± SD. *P < .05; **P < .01. (B) BCWM1 cells were treated with anti–miR-155 or scramble control for 48 hours. All of the BCWM1 cell samples were cultured and processed in parallel for total RNA extraction and GEP using Affymetrix GeneChip. Validated targets of miR-155 are shown in a heat map on the left and predicted targets of miR-155 are listed on the right. (C) qRT-PCR analysis of selected miR-155 targets in BCWM1 cells treated with anti–miR-155 or scramble control for 48 hours. Experiments were performed in triplicate and repeated 3 times. Data are shown as means ± SD. *P < .05; **P < .01. (D) Stable HEK293 cell lines overexpressing pre-miR-155 or empty vector as a control were generated by lentiviral infection. Cells were transiently transfected with the reporter plasmid pmirGLO driving luciferase expression with a target gene fragment containing the predicted miR-155 binding site or a mutated binding site as a control. Relative luciferase activity was decreased in miR-155–overexpressing HEK293 cells transfected with plasmids harboring miR-155 target sequences compared with plasmids harboring mutated target sequences. Experiments were performed in triplicate and repeated 3 times. Data are shown as means ± SD. *P < .05; **P < .01. (E) mRNA levels of miR-155 target genes were detected by qRT-PCR from normal donor blood CD19⁺ cells, BCWM1 and MEC1 cells, primary WM CD19⁺ cells, and primary CLL cells. Experiments were performed in triplicate and repeated 3 times. Data are shown as means ± SD. (F) mRNA levels of miR-155 target genes were detected by qRT-PCR from primary WM CD19⁺ cells treated with 20μM anti–miR-155 or scramble control for 48 hours. Experiments were performed in triplicate and repeated 3 times. Data are shown as means ± SD. (G) mRNA levels of miR-155 target genes were detected by qRT-PCR from primary CLL cells treated with 20μM anti–miR-155 or scramble control for 48 hours. Experiments were performed in triplicate and repeated 3 times. Data are shown as means ± SD.