Figure 2.

miR-142-3p regulates AC9 expression and cAMP production in CD4⁺CD25⁻ T cells and CD4⁺CD25⁺ regulatory T cells. (A) miR-142-3p targets 3′-UTR of AC9 messenger RNA (mRNA). miR-142-3p inhibited the expression of the luciferase gene containing AC9 3′-UTR but not mutated AC9 3′-UTR; ^*P<0.05, compared with AC9 3′-UTR group. (B) miR-142-3p regulates AC9 at the protein level. miR-142-3p inhibitor-transfected CD4⁺CD25⁻ T cells and miR-142-3p-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 detected by using real-time PCR (left). The relative level of AC9 protein was detected by Western blot (middle) and densitometric analysis (right). ^*P<0.05, compared with CD4⁺CD25⁻ T-cell control group (n=5 per group); ^#P<0.05, compared with CD4⁺CD25⁺ T-cell control group (n=5 per group). (C) miR-142-3p regulates the level of intracellular cAMP. cAMP was prepared from miR-142-3p inhibitor-transfected CD4⁺CD25⁻ T cells or miR-142-3p-transfected CD4⁺CD25⁺ T_REG cells 48 h after transfection, and measured using a cAMP-specific ELISA kit; ^*P<0.05, compared with control groups. 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; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; T_REG, regulatory T cells; UTR, untranslated region.