Identification of a miR-146b-Fas ligand axis in the development of neutropenia in T large granular lymphocyte leukemia

Abstract

Tlarge granular lymphocyte leukemia (T-LGLL) is characterized by the expansion of several large granular lymphocyte clones, among which a subset of large granular lymphocytes showing constitutively activated STAT3, a specific CD8⁺/CD4^- phenotype and the presence of neutropenia has been identified. Although STAT3 is an inducer of transcription of a large number of oncogenes, so far its relationship with miRNAs has not been evaluated in T-LGLL patients. Here, we investigated whether STAT3 could carry out its pathogenetic role in T-LGLL through an altered expression of miRNAs. The expression level of 756 mature miRNA was assessed on purified T large granular lymphocytes (T-LGLs) by using a TaqMan Human microRNA Array. Hierarchical Clustering Analysis of miRNA array data shows that the global miRNome clusters with CD8 T-LGLs. Remarkably, CD8 T-LGLs exhibit a selective and STAT3-dependent repression of miR-146b expression, that significantly correlated with the absolute neutrophil counts and inversely correlated with the expression of Fas ligand (FasL), that is regarded as the most relevant factor in the pathogenesis of neutropenia. Experimental evidence demonstrates that the STAT3-dependent reduction of miR-146b expression in CD8 T-LGLs occurs as a consequence of miR-146b promoter hypermethylation and results in the disruption of the HuR-mediated post-transcriptional machinery controlling FasL mRNA stabilization. Restoring miR-146b expression in CD8 T-LGLs lead to a reduction of HuR protein and, in turn, of FasL mRNA expression, thus providing mechanistic insights for the existence of a STAT3-miR146b-FasL axis and neutropenia in T-LGLL.

Figure 1

miRNome of T-LGLL patients correlates with STAT3 phosphorylation. Peripheral blood CD57⁺ T cells from T large granular lymphocyte leukemia (T-LGLL) patients (n=6) and healthy donors (HD, n=3) were collected. (A) Heatmap representation of the 756 miRNAs analyzed using the TaqMan microRNA array as described in the Methods. Results are expressed as arbitrary units (au) using U6 as reference control. (B) Whole-cell extracts (250×10³ cells) purified from the same cells as in (A) were loaded on gels and analyzed for STAT3-YP and total STAT3 expression level. Quantification of the relative normalized STAT3-YP protein levels are reported as arbitrary units (au) below the Western blot. Mean ± standard error of the mean (SEM) is shown, *P<0.05 by unpaired t-test. (C) Absolute neutrophil counts in each CD8 and CD4 T-LGLL patient (circle) and HD (triangle). Mean ± SEM is shown. **P<0.01 by unpaired t-test. Patients analyzed in each panel are specified in the Online Supplementary Table S5.

Figure 2

STAT3-dependent inhibition of miR-146b expression in CD8 T large granular lymphocytes. (A) miR-146b expression level in each CD8 (n=9) and CD4 (n=6) T large granular lymphocytes (T-LGLs) as well as in HD (n=9) together with mean±standard error of the mean (SEM) are shown. *P<0.05 by Kruskal-Wallis tests. (B) Correlation analysis between miR-146b expression and STAT3-YP (left, n=12), miR-146b expression and absolute neutrophil count (ANC) (central, n=18), and between ANC and STAT3-YP levels (right, n=12). Grey circles identify CD8 T-LGLs, empty triangles identify CD4 T-LGLs. miR-146b expression analyzed in real-time quatitative PCR (RT-qPCR) is reported as arbitrary units after U6 normalization. The relative STAT3-YP protein levels, normalized for total STAT3, are reported as arbitrary units (au). ANC is reported as cell×10⁹/L. Spearman correlation coefficient (p) and P-values are reported. (C) CD8 T-LGLs were cultured for 24 hours (h) in presence of 15 μM STATTIC or DMSO as control (n=7). miR-146b primary transcript (pri-miR-146b) expression, analyzed by RT-qPCR, is reported as Fold Induction (FI) relative to DMSO treated cells, after RPL32 normalization. Mean ± SEM are shown. ***P<0.001 by Wilcoxon signed-rank. Patients analyzed in each panel are specified in the Online Supplementary Table S5.

Figure 3

Promoter methylation prevents miR-146b expression in CD8 T large granular lymphocytes. (A) The methylation level of −687/−496, −149/+98 miR-146b promoter region and +44/+315 downstream region was analyzed by meDIP assay with anti-5meC antibody (Ab) in CD4 (n=3) and CD8 (n=3) T large granular lymphocyte leukemia (T-LGLs). Single value and mean±standard error of the mean (SEM) are reported as percentage over input. **P<0.01, ***P<0.001, not significant (ns) P>0.05 by unpaired t-test. (B) PBMCs from CD8 T-LGLL patients were cultured in presence of 2.5 μM 5-aza-2′-deoxycytidine (DAC) for three days (left) or 15 μM STATTIC for 24 hours (h) (right). pri-miR-146b (left) and DNMT1 (right) expression was analyzed. Data are expressed as Fold Induction (FI) relative to the untreated condition (DMSO). Mean±SEM of six (left panel) and seven (right panel) independent experiments is shown. *P<0.05, by Wilcoxon signed-rank test. Patients analyzed in each panel are specified in the Online Supplementary Table S5.

Figure 4

miR-146b controls Fas ligand mRNA expression targeting HuR. Correlation analysis between Fas ligand (FasL) mRNA expression and STAT3-YP levels (A; n=8), FasL mRNA expression and ANC (B; n=12), FasL mRNA expression and miR-146b expression levels (C; n=12) and soluble FasL (sFasL) and miR-146b expression levels (D; n=8). FasL mRNA is expressed as MNE relative to GAPDH, while miR-146b is expressed as au after U6 normalization. ANC is reported as cell×10⁹/L and sFasL is expressed in pg/mL. Spearman correlation coefficient (p) and P are reported. (E) FasL mRNA and FasL primary transcript (PT) were analyzed in CD8 (n=7) and CD4 (n=5) T-LGLs by real-time quatitative PCR (RT-qPCR). Data are expressed as MNE relative to GAPDH. Data are reported as mean ± standard error of the mean (SEM). *P<0.05, ns, not significant by Mann-Whitney U-test. Grey circles identify CD8 T-LGLs, empty triangles identify CD4 T-LGLs. Patients analyzed in each panel are specified in the Online Supplementary Table S5.

Figure 5

miR-146b regulates Fas ligand expression by targeting HuR in Jurkat cells. (A) Jurkat cells were transfected with 75pmol miR-scr or miR-146b. 48 hours (h) after transfection cells were processed and miR-146b and FasL mRNA expression were analyzed. Data are expressed as Mean Normalized Expression (MNE) relative to U6 (miR-146b) and GAPDH (FasL mRNA). Mean±standard error of the mean (SEM) (n=3) is shown. *P<0.05 by paired t-test. (B) Jurkat cells were transfected with 75pmol miR-scr or miR-146b. 48 h after transfection cells were processed and HuR protein level was analyzed by western blotting. One Western blot representative of two performed with similar results is shown. The relative HuR protein level, normalized for GAPDH, is reported as au below the Western blot. (C) Jurkat cells were transfected with 200pmol si-CTR or si-HuR and processed as described above. One Western blot representative of two performed with similar results is shown. Quantification of normalized HuR protein level and of FasL mRNA expression (Mean±SEM, n=2) in transfected Jurkat is shown below the Western blot.

Figure 6

HuR is the endogenous miR-146b target gene in CD8 T large granular lymphocytes. (A) CD8 T large granular lymphocytes (T-LGL) were transfected with 200 pmol miR-scr or miR-146b. 24 hours (h) after transfection cells were processed and miR-146b, HuR mRNA, FasL mRNA and primary transcript (PT) expression were analyzed by real-time quatitative PCR (RT-qPCR). Data are expressed as MNE relative to U6 (miR-146b) and RPL32 (HuR, FasL mRNA and PT). Mean±standard error of the mean (SEM) of two independent experiments is shown. (B) HuR protein level was analyzed by Western blot as described in the Methods in CD8 (n=5) and CD4 (n=5) whole-cell extracts (15 μg). Normalized HuR protein levels are reported as arbitrary units (au) below the Western blot. *P<0.01 by unpaired t-test. Patients analyzed in each panel are specified in the Online Supplementary Table S5.

Figure 7

Schematic representation of the pathogenic link between constitutively active STAT3, defective miR-146b expression and Fas ligand-mediated neutropenia, that specifically characterizes the CD8 subset of large granular lymphocyte leukemia. Methylation status of miR-146b promoter is represented as circles: empty circles mean not methylated cytosine, while black circles represent methylated cytosine.