microRNA 184 regulates expression of NFAT1 in umbilical cord blood CD4+ T cells

Abstract

The reduced expression of nuclear factor of activated T cells-1 (NFAT1) protein in umbilical cord blood (UCB)-derived CD4+ T cells and the corresponding reduction in inflammatory cytokine secretion after stimulation in part underlies their phenotypic differences from adult blood (AB) CD4+ T cells. This muted response may contribute to the lower incidence and severity of high-grade acute graft-versus-host disease (aGVHD) exhibited by UCB grafts. Here we provide evidence that a specific microRNA, miR-184, inhibits NFAT1 protein expression elicited by UCB CD4+ T cells. Endogenous expression of miR-184 in UCB is 58.4-fold higher compared with AB CD4+ T cells, and miR-184 blocks production of NFAT1 protein through its complementary target sequence on the NFATc2 mRNA without transcript degradation. Furthermore, its negative effects on NFAT1 protein and downstream interleukin-2 (IL-2) transcription are reversed through antisense blocking in UCB and can be replicated via exogenous transfection of precursor miR-184 into AB CD4+ T cells. Our findings reveal a previously uncharacterized role for miR-184 in UCB CD4+ T cells and a novel function for microRNA in the early adaptive immune response.

Trial registration: ClinicalTrials.gov NCT00003335.

Figures

Figure 1

Relative NFAT1 protein and mRNA expression in stimulated UCB and AB CD4+ T cells. (A) Approximately 2 × 106 isolated UCB and AB CD4+ T cells were stimulated in vitro as described for each of the designated time points and Western blot analysis performed. Data are representative of 5 independent experiments. (B) NFATc2 (NFAT1-encoding) mRNA was assayed in stimulated UCB and AB CD4+ T cells by qRT-PCR as described and normalized to UCB at 0 hours. Each bar indicates mean (± SEM) of 2 to 5 independent data points.

Figure 2

Diagram of the predicted NFATc2 3′ UTR/hsa-miR-184 interaction. The sequence of miR-184 was retrieved from the Sanger database and the sequences for NFATc2 were retrieved from NCBI Entrez Nucleotide Sequence Viewer. Diagram indicates position of the stop codon relative to the first indexed base of each transcript and the position of the predicted 3′ UTR base pairing region relative to the stop codon.

Figure 3

miR-184 in UCB CD4+ T cells. (A) miR-184 was quantified in AB (n = 8) and UCB (n = 10) samples by qRT-PCR as described. P value obtained from unpaired, 2-tailed Student t test. (B) miR-184 was quantified in UCB CD4+ T cells stimulated in vitro as described for the designated time points (representative of 3 experiments). (C) miR-184 was quantified in AB CD4+ T cells stimulated in vitro as described for the designated time points (representative of 2 experiments).

Figure 4

Interaction between the NFATc2 3′ UTR and miR-184. (A) PCR amplification of the predicted target region from UCB MNC and CD4+ cDNA. Sequences of primers are specified in the Methods section. (B) Expression of luciferase in transfected UCB CD4+ cells under the influence of minimal 3′ UTR (left column), the predicted miR-184 binding site from NFATc2 (middle column), or the cloned 3′ UTR from NFATc2 (right column) with antisense sequence to miR-184 or irrelevant control DNA sequence (representative of 3 independent experiments). (C) Expression of luciferase in transfected AB CD4+ cells under the influence of the aforementioned 3′ UTRs, exogenous pre-miR-184, and antisense to miR-184 (representative of 3 independent experiments). N.D. indicates data not determined. (D) Expression of luciferase in transfected UCB CD4+ cells under the influence of the cloned NFATc2 3′ UTR (left column), the same UTR with the predicted miR-184 seed region (4 nucleotides [nt]) removed (middle column), and the same UTR with the entire predicted miR-184 binding site removed (right column) with and without antisense to miR-184 (representative of 2 independent experiments). (E) Expression of luciferase in transfected AB CD4+ cells under the influence of the aforementioned 3′ UTRs, with and without precursor to miR-184 (representative of 2 independent experiments).

Figure 5

miR-184 negatively effects NFAT1 protein synthesis. (A) Quantification and representative blot of NFAT1 protein expression in UCB CD4+ T cells 16 hours after transfection with antisense to miR-184 (n = 3). (B) qRT-PCR analysis of samples in (A), confirming no significant change in NFATc2 mRNA quantity. (C) Quantification and representative blot of NFAT1 protein expression in AB CD4+ T cells under the following transfection with pre-miR-184 (n = 4). Blot bands were quantified using ImageJ software. (D) Western blot analysis of NFAT1 protein expression in AB CD4+ T cells 16 hours after transfection with antisense to miR-184.

Figure 6

IL2 transcription in stimulated CD4+ T cells. (A) IL2 transcription in stimulated UCB CD4+ T cells under the influence of miR-184 antisense. (B) IL2 transcription in stimulated AB CD4+ T cells after transfection of exogenous miR-184 precuror. Cells were stimulated in vitro 16 hours after transfection, and IL2 mRNA was assayed by qRT-PCR as described from 106 cells per data point. Data are representative of 2 independent experiments.