Fig. 1.
MicroRNA-31 (miR-31) is down-regulated in stimulated human T cells and impairs the balance of TH1/TH2 cytokine transcription. (A) CD3+ T cells were purified from peripheral blood of healthy donors (n = 4) and were left untreated or activated with anti-CD3/CD28 Dynabeads for 16, 24, 48, and 72 h, respectively. Relative expression of mature hsa-miR-31-5p was quantified by quantitative polymerase chain reaction (qPCR) with U47 RNA as endogenous control. Data are means ± SD; *P = 0.038, 48 versus 0 h, and P = 0.036, 72 versus 0 h; §P = 0.0395, 48 versus 16 h, and P = 0.0168, 72 versus 16 h, in one-way ANOVA with Tukey post test. (B) Left panel presents relative SUZ12 and EZH2 mRNA levels at the indicated time points, as determined by qPCR in stimulated human T cells from A. Data are means ± SD; SUZ12: not significant (ns) P = 0.2954, 16 versus 24 h, *P = 0.0107, 24 versus 0 h, and P = 0.0352, 16 versus 0 h as well as EZH2: ns P = 0.9811, 16 versus 24 h, *P = 0.0022, 24 versus 0 h, and **P = 0.0008, 16 versus 0 h in one-way ANOVA with Tukey post test. Right panel depicts epigenetic regulation of miR-31 by polycomb repressive complex 2 (PRC2), consisting of the catalytic subunit EZH2 and three noncatalytic subunits SUZ12, RBAP48, and embryonic ectoderm development (latter two not shown). This complex is recruited to a genomic region (PRC response element [PRE]) upstream the miR-31 locus on chromosome 9 (Chr 9p21.3), leading to miR-31 repression via chromatin silencing through histone H3 methylation (Met). (C, D) CD4+ T cells from healthy donors were transiently transfected with Cy3-labeled pre-miR scrambled control, Ambion pre-miR-31-5p, or pre-miR scrambled control, respectively, and stimulated for 24 h with anti-CD3/CD28 Dynabeads. (C) Transfection efficiency was determined by flow cytometry as the percentage of Cy3-fluorescent cell number in total cell number. Left panel shows representative histogram, and right panel indicates calculated total transfection efficiency (open bar) of n = 3 individual experiments performed in triplicates. Data are means ± SD. (D) Total RNA was isolated from stimulated CD4+ T cells transiently transfected with Ambion pre-miR-31-5p or scrambled control. Relative interferon (IFN)-γ, interleukin (IL)-2, and IL-4 mRNA expression was measured by quantitative real-time PCR. Data represent means ± SD of n = 5 healthy donors. IFN-γ: *P = 0.001, IL-2: *P = 0.005, and IL-4: **P = 0.0001 in paired Student’s t test.

MicroRNA-31 (miR-31) is down-regulated in stimulated human T cells and impairs the balance of TH1/TH2 cytokine transcription. (A) CD3+ T cells were purified from peripheral blood of healthy donors (n = 4) and were left untreated or activated with anti-CD3/CD28 Dynabeads for 16, 24, 48, and 72 h, respectively. Relative expression of mature hsa-miR-31-5p was quantified by quantitative polymerase chain reaction (qPCR) with U47 RNA as endogenous control. Data are means ± SD; *P = 0.038, 48 versus 0 h, and P = 0.036, 72 versus 0 h; §P = 0.0395, 48 versus 16 h, and P = 0.0168, 72 versus 16 h, in one-way ANOVA with Tukey post test. (B) Left panel presents relative SUZ12 and EZH2 mRNA levels at the indicated time points, as determined by qPCR in stimulated human T cells from A. Data are means ± SD; SUZ12: not significant (ns) P = 0.2954, 16 versus 24 h, *P = 0.0107, 24 versus 0 h, and P = 0.0352, 16 versus 0 h as well as EZH2: ns P = 0.9811, 16 versus 24 h, *P = 0.0022, 24 versus 0 h, and **P = 0.0008, 16 versus 0 h in one-way ANOVA with Tukey post test. Right panel depicts epigenetic regulation of miR-31 by polycomb repressive complex 2 (PRC2), consisting of the catalytic subunit EZH2 and three noncatalytic subunits SUZ12, RBAP48, and embryonic ectoderm development (latter two not shown). This complex is recruited to a genomic region (PRC response element [PRE]) upstream the miR-31 locus on chromosome 9 (Chr 9p21.3), leading to miR-31 repression via chromatin silencing through histone H3 methylation (Met). (C, D) CD4+ T cells from healthy donors were transiently transfected with Cy3-labeled pre-miR scrambled control, Ambion pre-miR-31-5p, or pre-miR scrambled control, respectively, and stimulated for 24 h with anti-CD3/CD28 Dynabeads. (C) Transfection efficiency was determined by flow cytometry as the percentage of Cy3-fluorescent cell number in total cell number. Left panel shows representative histogram, and right panel indicates calculated total transfection efficiency (open bar) of n = 3 individual experiments performed in triplicates. Data are means ± SD. (D) Total RNA was isolated from stimulated CD4+ T cells transiently transfected with Ambion pre-miR-31-5p or scrambled control. Relative interferon (IFN)-γ, interleukin (IL)-2, and IL-4 mRNA expression was measured by quantitative real-time PCR. Data represent means ± SD of n = 5 healthy donors. IFN-γ: *P = 0.001, IL-2: *P = 0.005, and IL-4: **P = 0.0001 in paired Student’s t test.

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