miR-135b targets FOXO1 and regulates chemosensitivity. (A) Structure of TuD RNA expression vector. TuD RNA for miR-135b contains miRNA-binding site (MBS) that is partially complementary to miR-135b. Details of TuD RNA structure have been described previously.¹¹  (B) Introduction of TuD RNA against miR-135b into ALCL cells. Flow cytometry profiles indicating transduction efficiency in Karpas 299 cells. FSC indicates forward scatter. (C) Inhibition of miR-135b function by TuD RNA for miR-135b. Left and right panels show qRT-PCR result and luciferase assay monitoring miR-135b activity, respectively. NC indicates negative control. (D) Sequence alignment between miR-135b and its putative binding site in the FOXO1 3′UTR. (E) miR-135b targets FOXO1. HEK293T cells were transfected with luciferase reporter containing the FOXO1 3′UTR with wild-type or mutated target site (shown in panel D), along with empty vector, miRNA-lacZ expression control vector (miRNA for lacZ), or pri-miR-135b expression vector [miR-135b (+)]. Luciferase assay was performed 48 hours after transfection (***P < .001). (F) Suppression of FOXO1 protein level by miR-135b. HeLa cells were transiently transfected with miR-135b and subjected to immunoblot analysis. (G) Elevated expression of FOXO1, p21, and p27 by miR-135b inhibition in ALCL cells. SUDHL-1 and SUP-M2 cells were infected with lentivirus harboring TuD-NC or TuD-miR-135b and applied to immunoblot analysis. The quantification results were shown in the bottom panel. (H) miR-135b–mediated attenuation of chemosensitivity. Jurkat cells with empty or pri-miR-135b vectors were treated with cytosine β-d-arabinofuranoside (AraC), followed by the assessment of cell viability (left) and apoptosis (right; *P < .05; ***P < .001; n.s., not significant).