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. 2022 Mar 28;23(5):e53475. doi: 10.15252/embr.202153475

Figure 6. miPEP31 induces Treg‐cell differentiation through inhibiting miR‐31 expression.

Figure 6

  • A
    miR‐31 expression in anti‐CD3/28‐activated CD4+ T cells treated by different dose of synthetic miPEP31. Data in (A) are presented as mean ± SEM of four biological replicates. ***P < 0.001, ****P < 0.0001 (0 μM vs. 0.1 μM or 1 μM or 10 μM), nonparametric one‐way ANOVA, and Kruskal–Wallis test.
  • B
    qPCR analysis of the expression of pri‐miR‐31 in anti‐CD3/28‐activated CD4+ T cells treated by different doses of miPEP31. Data in (B) are presented as mean ± SEM of four biological replicates. *P < 0.05, ***P < 0.001 (0 μM vs. 0.1 μM or 1 μM or 10 μM), nonparametric one‐way ANOVA, Kruskal–Wallis test.
  • C
    Anti‐CD3/28‐activated CD4+ T cells were treated by 10 μM miPEP31 or scPEP, the expression of miR‐31, miR‐146a, miR‐18a, miR‐203, and miR‐204 was detected by qPCR. Results are presented as the ratio of miRNA to the small nuclear RNA U6. Data in (C) are presented as mean ± SEM of four biological replicates. ns, not significant, ***P < 0.001 (scPEP vs. miPEP31), nonparametric one‐way ANOVA, Kruskal–Wallis test.
  • D, E
    Flow cytometric analysis of Treg cells polarized from naive CD4+ T cells of miR‐31fl/fl‐CD4‐Cre (cKO) mice in the presence of 10 μM synthetic scPEP or miPEP31. Data in (E) are presented as mean ± SEM of six biological replicates. ns, not significant, unpaired two‐tailed Student’s t‐test.
  • F
    Clinical scores of scPEP‐ or miPEP31‐treated cKO or WT mice after the induction of EAE were assessed every day. scPEP or miPEP31 was injected intravenously into mice every 3 days starting from day 8 postimmunization. Data in (F) are presented as mean ± SEM of three independent experiments with 9 to 10 mice per group in each. ns, not significant, ****P < 0.0001, repeated measures two‐way ANOVA and Tukey post‐hoc test.
  • G, H
    CD4+ T cells were transduced with pCDNA 3.1‐NC or pCDNA 3.1‐pri‐miR‐31 and cultured in Treg polarizing condition in the presence of 10 μM synthetic scPEP or miPEP31 for 3 days. The Treg differentiation was analyzed by flow cytometry. Data in (H) are presented as mean ± SEM of four biological replicates. ns, not significant, ***P < 0.001, unpaired two‐tailed Student’s t‐test.