Identification of NPM-ALK-STAT3-miR-135b axis in ALCL. (A) Induction of LEMD1/miR-135b by NPM-ALK. Jurkat cells were transduced with lentivirus carrying NPM-ALK or kinase-dead NPM-ALK (K210R) and subjected to qRT-PCR analysis. (B) NPM-ALK/STAT3–dependent up-regulation of LEMD1/miR-135b in ALCL. Effects of shRNAs against NPM-ALK and STAT3 were evaluated in SUDHL-1 cells (*P < .05; **P < .01). (C) Up-regulation of LEMD1/miR-135b by ca-STAT3. Jurkat cells were transduced with lentivirus carrying mouse ca-STAT3 and subjected to qRT-PCR analysis. (D) STAT3 binding sites in LEMD1 genomic region. Top panel indicates schematic genomic organization of human LEMD1 gene and miR-135b. Sequence conservation between human and mouse is represented as the percentage of conservation in the Vista analysis shown in the middle panel. We analyzed putative STAT3-binding sites within the conserved region in ChIP analysis, and we found that STAT3 bound to 3 sites in the bottom panel, as shown in panel E. Number 1 site (TTAAGGGAA) is conserved between human and mouse. (E) Binding of STAT3 to LEMD1 genomic regions analyzed by ChIP analysis in SUP-M2 (left) and Jurkat (right) cells. Total chromatin before immunoprecipitation was used as positive control for PCR. Jurkat cells were used as negative control. (F) Enhanced miR-135b activity in ALCL. Cells were transfected with miRNA sensor vectors and applied to luciferase assay. Reflecting high miR-135b expression, luciferase expression levels from miR-135b sensor vector were remarkably lower than those from control sensor vector in ALCL cells but not in Jurkat cells.