Negative regulation of miR-150 against autocrine IL-22-CCL20-CCR6 pathway in CTCL cells. (A) qPCR of IL-22 and IL-17A in CD4⁺ T cell (n = 14) and advanced CTCL samples (n = 12). P values were calculated using the Mann-Whitney U test. Asterisks (*) indicate statistical significance: **.001 ≤ P < .01; ***P < .001. Bars are means ± SEM of 3 independent experiments. (B) Western blot of IL-22RA1 and CCR6 in 5 normal CD4⁺ T cell (CD4-1 to CD4-5), My-La and HH cell lines, and 5 primary CTCL cases. Jeko-1 (mantle cell lymphoma cell line) is positive control of IL-22RA1.³⁸  (C) ELISA of IL-10, IL-17, IL-22, and CCL20 in CTCL cell lines. Cells (1 × 10⁴) were cultured for 24 hours or 48 hours in 48-well dishes. Each well contained 500 μL of culture medium supplemented with 2% HS or HS free [HS(-)]. Y-axis: pg/mL. ELISA of CCL20 in CTCL cell lines. (D) Effect of IL22RA1 knockdown on IL-22 and CCL20. Left: expression of IL22RA1 in CTCL cell lines transiently transfected with siIL22RA1(744 and 1571). Right: western blot analysis of IL22RA1 in CTCL cells transiently transduced with siIL22RA1(1571) and control [+siSC(scrambled)]. Tubulin served as a control for IL22RA1. ELISA of IL-22 and CCL20 in CTCL cells transiently transduced with siIL22RA1(1571). These cells were cultured for 12 hours or 24 hours. P values were calculated using Student t test. Asterisks (*) indicate statistical significance: *.01 ≤ P < .05; **.001 ≤ P < .01; ***P < .001. Bars are means ± SEM of 6 independent experiments. (E) ELISA of IL-22 and CCL20 in GFP-empty (control) or GFP-miR-150–transduced CTCL cells transiently transduced with siCCR6 (shown as “+siCCR6”). ELISA of IL-22 and CCL20 in GFP-siCCR6–transduced CTCL cells are also shown. These cells were cultured for 24 hours or 48 hours. Asterisks (*) indicate statistical significance: **.001 ≤ P < .01. Bars are means ± SEM of 3 independent experiments. NS: not significant.