Identification of novel microRNA signatures linked to acquired aplastic anemia

Abstract

Emerging evidence indicates that microRNA control and modulate immunity. MicroRNA have not been investigated in acquired aplastic anemia, a T-cell-mediated immune disease. Analysis of 84 microRNA expression levels in CD4(+) and CD8(+) T cells of patients with aplastic anemia revealed concurrent down-regulation of miR-126-3p, miR-145-5p, miR-223-3p, and miR-199a-5p (>3-fold change, P<0.05) in both T-cell populations, which were unique in aplastic anemia compared to other hematologic disorders. MiR-126-3p and miR-223-3p were down-regulated in CD4(+) T effector memory cells, and miR-126-3p, miR-145-5p, and miR-223-3p were down-regulated in CD8(+) T effector memory and terminal effector cells. Successful immunosuppressive therapy was associated with restoration to normal expression levels of miR-126-3p, miR-145-5p, and miR-223-3p (>2-fold change, P<0.05). In CD4(+) and CD8(+) T cells in aplastic anemia patients, MYC and PIK3R2 were up-regulated and proved to be targets of miR-145-5p and miR-126-3p, respectively. MiR-126-3p and miR-145-5p knockdown promoted proliferation and increased interferon-γ and granzyme B production in both CD4(+) and CD8(+) T cells. Our work describes previously unknown regulatory roles of microRNA in T-cell activation in aplastic anemia, which may open a new perspective for development of effective therapy. Clinicaltrials.gov identifier: NCT 01623167.

Trial registration: ClinicalTrials.gov NCT01623167.

Copyright© Ferrata Storti Foundation.

Figures

Figure 1.

Distinct miRNA expression patterns in CD4+ and CD8+ T cells of AA patients. Volcano plots of results from 84 miRNA known to be involved in lymphocyte activation in (A) CD4+ and (C) CD8+ T cells (AA patients, n=12; healthy donors, n=8) and (E) CD19+ B cells (AA patients, n=9; healthy donors, n=7) using miRNA PCR-array. The x-axis is the estimated difference in expression measured in log2; vertical lines refer to a 3-fold difference in expression between the two groups. MiRNA highly expressed in AA or healthy donors are on the right or the left, respectively. The y-axis is the significance of the difference measured in −log10 of the P-value; the horizontal line represents our cutoff for significance at P<0.01. Hierarchical clustering of miRNA in (B) CD4+ T cells, (D) CD8+ T cells, or (F) CD19+ B cells was visualized by heatmap analysis. A red-blue color scale depicts normalized miRNA expression levels in Ct values (red: high, blue: low). (G) Venn diagram presentation of PCR array data.

Figure 2.

Comparison of expression levels of specific miRNA in other hematologic diseases. RT-qPCR analysis of miR-126-3p, miR-145-5p, miR-199a-5p, and miR-223-3p expression in (A) CD4+ and (B) CD8+ T cells from AA (n=12), low-risk MDS (n=5), and SCD (n=5) patients, and healthy controls (n=8). MiRNA relative expression was obtained by normalizing to RNU-2 expression. *P<0.05 [two-way analysis of variance (ANOVA)].

Figure 3.

MiRNA expression in CD4+ and CD8+ T cell subsets of AA patients. RT-qPCR analysis of miR-126-3p, miR-145-5p, miR-199a-5p, and miR-223-3p expression in (A) CD4+ and (B) CD8+ T cell subsets from AA patients (n=3) and healthy controls (n=3). The TE population in CD4+ T cells was not examined, due to the low number of cells in healthy controls. Relative expression of miRNA was calculated by normalizing to RNU-2 expression. *P<0.05 [two-way analysis of variance (ANOVA)].

Figure 4.

MiRNA expression changes after immunosuppressive therapy (IST). RT-qPCR analysis of miR-126-3p, miR-145-5p, miR-199a-5p, and miR-223-3p expression in (A) CD4+ and (B) CD8+ T cells from AA patients at onset (n=6) and after IST (n=6). Relative expression of miRNA was calculated with respect to RNU-2 expression. Relative expression levels were normalized to those of healthy controls. *P<0.05 (Student t-test).

Figure 5.

MiRNA target gene prediction and validation in CD4+ and CD8+ T cells. Volcano plots represent relative expression levels of 84 mRNA analyzed using miRNA Targets PCR Arrays. These 84 mRNA were predicted or experimentally validated to be targets of four miRNA (miR-126-3p, miR-145-5p, miR-199a-5p, and miR-223-3p) in (A) CD4+ and (G) CD8+ T cells from AA patients (n=6) and healthy donors (n=6). The x-axis is estimated difference in expression measured in log2; vertical lines refer to a 1.5-fold difference in expression between the two groups. mRNA highly expressed in AA or healthy donors are on the right or the left, respectively. The y-axis is the significance of the difference measured in −log10 of a P-value; the horizontal line indicates our cutoff for significance at P<0.05. RT-qPCR analysis of several target gene expression in (B) CD4+ or (H) CD8+ T cells from AA patients (n=12) and healthy controls (n=8) *P<0.05 [two-way analysis of variance (ANOVA)]. After treatment of CD4+ T cells with anti-miR-145-5p, relative expression of (C) miR-145-5p or (D) MYC was measured by RT-qPCR 24 h later while (E) MYC protein expression was assessed by immunoblot analysis 48 h later. After treatment of CD8+ T cells with anti-miR-126-3p, relative gene expression of (I) miR-126-3p or (J) PIK3R2 and (K) protein expression of PIK3R2 were measured in similar manners. (F, L) Dual luciferase reporter assay. The relative luciferase activity of MYC 3′UTR or PIK3R2 3′UTR construct was measured by co-transfection with miR-145-5p in CD4+ T cells or miR-126-3p in CD8+ T cells, respectively. Data are shown as percentages of controls (cells co-transfected with MYC 3′UTR or PIK3R2 3′UTR construct and control miRNA, respectively). The relative activity of firefly luciferase expression was normalized to renilla luciferase activity. Relative RNA expression of miRNA or mRNA was calculated by normalizing to RNU-2 or β-actin expression, respectively. Data are from three independent experiments (means ± SEM). *P<0.05 (Student t-test).

Figure 6.

Functional study of miRNA down-regulated in T cells of AA patients. Purified CD4+ and CD8+ T cells from healthy controls were labeled with CFSE and transfected with anti-miR-126-3p and/or anti-miR-145-5p, followed by stimulation with anti-CD3/CD28 beads. (A) Representative histograms of CD4+ and CD8+ T-cell proliferation. (B) Frequency of CD4+ or CD8+ T-cell proliferation was quantified as the median fluorescence intensity (MFI) of CFSE after transfection of control, anti-miR-126-3p, anti-miR-145-5p, or both miRNA. *P<0.05 [one-way analysis of variance (ANOVA)]. (C) Percentages of TN, TCM and TEM were examined in CD4+ or CD8+ T cells transfected with anti-miR-126-3p and/or anti-miR-145-5p 48 h later. *P<0.05 [one-way analysis of variance (ANOVA)]. (D) Representative plots of intracellular GZMB and IFN-γ expression following in vitro stimulation with anti-CD3/CD28 beads. (E) Percentages of T cells producing GZMB, IFN-γ, and IL-2 were examined in CD4+ or CD8+ T cells transfected with anti-miR-126-3p and/or anti-miR-145-5p 48 h later. Data are from three independent experiments (means ± SEM). *P<0.05. [two-way analysis of variance (ANOVA)].

Figure 7.

Overexpression of miR-126-3p and miR-145-5p in T cells from AA patients decreases T-cell proliferation and inhibits IFN-γ production. Purified CD4+ and CD8+ T cells from AA patients were labeled with CFSE and transfected with miR-126-3p and/or miR-145-5p, followed by stimulation with anti-CD3/CD28 beads. (A) Representative histograms of CD4+ and CD8+ T-cell proliferation. (B) Frequency of CD4+ or CD8+ T-cell proliferation was quantified as the median fluorescence intensity (MFI) of CFSE from dividing cells, after transfection of control, miR-126-3p, miR-145-5p, or both miRNA. *P<0.05 [one-way analysis of variance (ANOVA)]. (C) Representative plots of intracellular IFN-γ expression following in vitro stimulation with anti-CD3/CD28 beads. (D) Percentages of T cells producing GZMB, IFN-γ, and IL-2 were examined in CD4+ or CD8+ T cells transfected with miR-126-3p and/or miR-145-5p 48 h later. Data are from three independent experiments (means ± SEM). *P<0.05. [two-way analysis of variance (ANOVA)].