Page 197 - Haematologica May 2020

miR-146b in T-LGL Leukemia patients
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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 expres- sion 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 cellx109/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.
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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.
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