Figure 4.

FOXP3 regulates cAMP production by regulating miR-142-3p and AC9. (A,B) FOXP3 negatively regulates miR-142-3p. FOXP3 messenger RNA (mRNA) and miR-142-3p in CD4⁺CD25⁺ T_REG cells transfected with (A) FOXP3 small interfering RNA (siRNA) or (B) in CD4⁺CD25⁻ T cells transfected with FOXP3-expressing vector, were detected by real-time RT–PCR. The miR-142-3p level in the control group was designated as 1. (C) FOXP3 increases AC9 expression. pFOXP3-transfected CD4⁺CD25⁻ T cells and FOXP3 siRNA-transfected CD4⁺CD25⁺ T cells were used for mRNA analysis 48 h after transfection, and for protein analysis 72 h after transfection. AC9 mRNA was determined by real-time RT–PCR (upper), and AC9 protein was determined by Western blot (lower). (D) FOXP3 regulates the level of cytosolic cAMP. cAMP was prepared from FOXP3-expressing vector-transfected CD4⁺CD25⁻ T cells (left) or FOXP3 siRNA-transfected CD4⁺CD25⁺ T_REG cells (right) 48 h after transfection, and measured using the cAMP-specific ELISA kit; ^*P<0.05, compared with control. The data in this figure are the representatives of two independent experiments (n=6 per group in each experiment). AC9, adenylyl cyclase 9; cAMP, cyclic AMP; ELISA, enzyme linked immunosorbent assay; FOXP3, forkhead box P3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription PCR; T_REG, regulatory T cells.