Figure 4
CCR6 is a potential target of miR-150 in CTCL cells. (A) Detection of potential miR-150 target genes in GFP-miR-150 or GFP-empty (control) transduced CTCL cell lines. Left (downregulation): heat map of genes showing a GFP-miR-150 (shown as “miR-150”)/GFP-empty (shown as “control”) ratio <0.75. Right (upregulation): genes showing miR-150/control ratio >1.3. (B) CCR6 expression of normal CD4+ T cells (n = 14), CTCLs (10 cases of advanced MF and 2 cases of SzS). P values were calculated using the Mann-Whitney U test. Asterisks (*) indicate statistical significance: **.001 ≤ P < .01. (C) Immunohistochemistry of CCR6, CD4, and HE in primary CTCL (CTCL1 and CTCL3). (D) Taqman PCR analysis of CCR6 in the cells transfected with GFP-miR-150 or GFP-control. Y-axis: relative expression of CCR6 adjusted in GFP-transfected cells (2−ΔΔCt). 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 3 independent experiments. (E) Western blot analysis of Notch 3, c-Myb, CCR6, and tubulin in indicated transfected cell lines. Fold changes in CCR6 levels are shown below the gels. Control: GFP-empty (shown as “control”) transduced cells; miR-150: GFP-miR-150 transduced cells. Tubulin served as a control of western blot analysis. (F) Immunohistochemistry of CCR6 in My-La and HH cells transfected with or GFP-empty control (shown as “control”) or GFP-miR-150 (shown as “miR-150”). (G) Luciferase reporter assays showing CCR6 expression in Rat-1 fibroblasts transfected with miR-16-1, miR-29a, miR-150, or PMXs-puro control vector. Blots showing miR-150 expression are beside the bars: pMIR, empty vector (pMIR vector); Mut, CCR6 3′UTR mutation-inserted pMIR vector; and Wt, CCR6 3′UTR wild-type–inserted vector. P values were calculated using Student t test. Asterisks (*) indicate statistical significance: **.001 ≤ P < .01. Bars are means ± SEM of 3 independent experiments. (H) Western blot analysis of CCR6 and tubulin in My-La and HH cells transfected with GFP-control (control), GFP-miR-16-1 (miR-16), GFP-miR-29a (miR-29a), or GFP-miR-150 (miR-150).

CCR6 is a potential target of miR-150 in CTCL cells. (A) Detection of potential miR-150 target genes in GFP-miR-150 or GFP-empty (control) transduced CTCL cell lines. Left (downregulation): heat map of genes showing a GFP-miR-150 (shown as “miR-150”)/GFP-empty (shown as “control”) ratio <0.75. Right (upregulation): genes showing miR-150/control ratio >1.3. (B) CCR6 expression of normal CD4+ T cells (n = 14), CTCLs (10 cases of advanced MF and 2 cases of SzS). P values were calculated using the Mann-Whitney U test. Asterisks (*) indicate statistical significance: **.001 ≤ P < .01. (C) Immunohistochemistry of CCR6, CD4, and HE in primary CTCL (CTCL1 and CTCL3). (D) Taqman PCR analysis of CCR6 in the cells transfected with GFP-miR-150 or GFP-control. Y-axis: relative expression of CCR6 adjusted in GFP-transfected cells (2−ΔΔCt). 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 3 independent experiments. (E) Western blot analysis of Notch 3, c-Myb, CCR6, and tubulin in indicated transfected cell lines. Fold changes in CCR6 levels are shown below the gels. Control: GFP-empty (shown as “control”) transduced cells; miR-150: GFP-miR-150 transduced cells. Tubulin served as a control of western blot analysis. (F) Immunohistochemistry of CCR6 in My-La and HH cells transfected with or GFP-empty control (shown as “control”) or GFP-miR-150 (shown as “miR-150”). (G) Luciferase reporter assays showing CCR6 expression in Rat-1 fibroblasts transfected with miR-16-1, miR-29a, miR-150, or PMXs-puro control vector. Blots showing miR-150 expression are beside the bars: pMIR, empty vector (pMIR vector); Mut, CCR6 3′UTR mutation-inserted pMIR vector; and Wt, CCR6 3′UTR wild-type–inserted vector. P values were calculated using Student t test. Asterisks (*) indicate statistical significance: **.001 ≤ P < .01. Bars are means ± SEM of 3 independent experiments. (H) Western blot analysis of CCR6 and tubulin in My-La and HH cells transfected with GFP-control (control), GFP-miR-16-1 (miR-16), GFP-miR-29a (miR-29a), or GFP-miR-150 (miR-150).

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