Figure 6: Mir-125a inhibits several effector T lineage factors.

(a) MiR-125a targets Stat3, Il13 and Ifng. Dual luciferase assay of HEK293T cells co-transfected with reporter vectors containing the 3′UTRs of different genes and miR-125a agomir or control agomir. The renilla luciferase activity was normalized to the Firefly luciferase activity. Data are shown as relative luciferase activities of cells transfected with miR-125a related to those of the control cells. (b) Comparison of miR-125a-responsive elements in human and mice transcripts encoding Stat3 (STAT3 and Stat3, respectively), Il13 (IL13 and Il13, respectively) and Ifng (IFNG and Ifng, respectively) by TargetScan. Colours in the key indicate branch lengths (and match to miR-125a). CDS, coding sequence. (c) Dual luciferase assay of HEK293T cells co-transfected the indicated WT or point-mutated 3′UTR-driven reporter constructs (wt UTR or mut UTR, respectively) with miR-125a or control agomirs. (d) Western blotting of endogenous STAT3 protein levels after transfection of HEK293T cells or infection of CD4⁺ T cells with different doses of miR-125a agomirs or miR-125a retrovirus for 2 days. MiR-148a was used as a control. (e) Enzyme-linked immunosorbent assay examination of IFN-γ and IL-13 in the supernatants of CD4⁺ T cells transfected with retrovirus-based miR-125a or empty vector, and differentiated under T_H1- or T_H2-skewing conditions for 4 days. (f) Quantitative RT–PCR analysis of the mRNA levels of Stat3, Il13 and Ifng after the retrovirus-based overexpression of miR-125a in CD4⁺ T cells for 4 days. The relative expression levels were normalized to the expression of mouse Rpl13a. Data are representative of four (a) or three (c–f) independent experiments (mean±s.e.m.). *P<0.05, **P<0.01 versus control (two-tailed Student’s t-test; a,e,f) or (one-way analysis of variance and least significant difference t-test; c).