Tax triggers ORP4L expression and T-cell malignant transformation via suppression of miR-31. (A-B) qPCR (A) and western blot (B) analyses confirming the effects of miR-31 inhibitor on ORP4L expression in HeLa cells and the effects of miR-31 mimic on ORP4L expression in MT-4 cells. Mean ± standard deviation (SD; n = 3 experimental repeat; Student t test). (C) qPCR analysis of miR-31 expression in T cells of patients with ATL (n = 37), as compared with normal T cells (n = 5). (D) Pearson’s correlation coefficient of miR-31 and ORP4L expression within human ATL specimens (n = 37). (E) Multicolor fluorescence in situ hybridization analysis of the genomic status of miR-31 encoding (green probe) and its host gene (origin probe) regions in T cells of 2-month-old WT and LCK/R26^Tax mice. The chromosome 4 centromere (red probe) is included as a control. Scale bar, 5 μm. (F) miR-31 expression in 2-month-old WT and LCK/R26^Tax mouse T cells analyzed by qPCR. Mean ± SD (n = 3 mice of each group; Student t test). (G) Western blot analysis of the expression of ORP4L in LCK/R26^Tax T cells or LCK/R26^Tax T cells overexpressing miR-31. The cells were isolated and infected with lentivirus carrying control RNA or miR-31 and cultured for 96 hours in vitro. (H) Western blot analysis of the expression of ORP4L in human normal T cells or human ATL T cells overexpressing miR-31. The cells were isolated and infected with lentivirus carrying control RNA or miR-31 and cultured for 96 hours in vitro. (I) Kaplan-Meier comparative survival analysis of B-NDG mouse recipients of WT T cells or LCK/R26^Tax T cells overexpressing miR-31 or overexpressing both miR-31 and ORP4L. The LCK/R26^Tax T cells were coinfected with the lentivirus carrying control RNA miR-31, empty vector (Emp.vec.), or ORP4L as follows: control RNA+Emp.vec., miR-31+Emp.vec., miR-31+ORP4L, and cultured for 96 hours in vitro before transplantation (n = 7; mice of each group, log-rank test). (J) Representative images of peripheral blood smears from B-NDG mice treated as in panel I. The number of abnormal lymphocytes on smears in each cohort are indicated below the images. Scale bar, 10 μm. (K) The percentage of CD3⁺CD4⁺, CD4⁺CD8⁺, CD44⁺CD25⁺, and CD3⁺c-Kit⁺ cells in peripheral blood of B-NDG mice treated as in panel I. Mean ± SD (n = 7 mice of each group; Student t test). (L) Representative histologic hematoxylin and eosin– and anti-CD3–stained sections showing T-cell infiltration in spleen and liver of B-NDG mice treated as in panel I. Scale bars, 100 μm. (M) Kaplan-Meier comparative survival analysis of B-NDG mouse recipients of T cells of patients with ATL (ATL#21). The cells were isolated and coinfected with lentivirus carrying control RNA, miR-31, Emp.vec. or ORP4L as follows: control RNA+Emp.vec., miR-31+Emp.vec., control RNA+ORP4L, and miR-31+ORP4L and cultured for 96 hours in vitro before transplantation (n = 8 mice each group, log-rank test). (N) The percentage of human CD45⁺CD3⁺ ATL T cells in peripheral blood of B-NDG mice treated as in panel M. Mean ± SD (n = 8 mice in each group; Student t test). ***P < .001.