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. 2019 Mar 19;10:524. doi: 10.3389/fimmu.2019.00524

Figure 4.

Figure 4

PRMT5-targeted miRNAs are downregulated after memory human Th cell activation. (A–D) Human memory Th1 (A,B) or Th2 (C,D) cells were activated with anti-CD3/CD28 and PRMT5 protein expression (A,C, western blot) and PRMT5 mRNA transcripts (B,D, Real-Time PCR) were analyzed at the indicated timepoints. Resting cells correspond to Th1/Th2 cells (after two rounds of differentiation) and 7 days after last stimulation. (E) Human PRMT5 3′UTR and predicted miRNA-binding sites. The miRNA sequence is shown below the PRMT5 mRNA sequence (straight lines and dotted lines connecting the mRNA and miRNA nucleotides indicate Watson-Crick base pairing and wobble base pairing, respectively). Bolded nucleotides indicate the seed sequence. (F) Cos-7 cells were transfected with pmiRGlo Dual Glo Luciferase plasmid containing human PRMT5 3′UTR and miR-15a, miR-15b, miR-96, miR-16, miR-140-3p, miR-146a, miR-146b miR-195, miR-322/424 (G) Cos-7 cells were transfected with pmiRGlo Dual Glo Luciferase plasmid containing human WT or mutated PRMT5 3′UTR and miR-140-3p. Firefly luciferase expression was normalized to Renilla luciferase expression and expressed as a ratio of Luc/RLuc relative to nonsense (NS) control. (H,I) miR-140-3p expression was analyzed by real time PCR in Th1 (H) and Th2 (I) cells after T cell activation. Data are representative of three independent experiments. Error bars represent SD. One-way ANOVA, followed by Dunnett's multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.