Fig. 4

The DENN/MADD gene is a direct downstream target of miR-24-3p: validation by luciferase assays and Western blot: (A) a human DENN/MADD 3′-UTR fragment containing a WT or MT miR-24-3p binding sequence was cloned downstream of the luciferase reporter gene. The plasmids were transfected into K562 and HL62 cells with or without miR-24-3p mimics. Renilla luciferase plasmids were cotransfected for normalization. A pGL3-control vector was cotransfected as a positive control. Analysis of luciferase activity: The data from the reporter assays are presented as the mean ± SEM from three independent experiments performed in triplicate (K562***p < 0.0005) (HL62, ***p < 0.0006). B Western blot and qPCR analysis of DENN/MADD expression after HD CD3 T-cell transduction by LV-miR-24-3p or lenti-CTRL and AML CD3 T-cell transduction by the antagomiR-miR-24-3p-GFP or lenti-CTRL construct showed that miR-24-3p reduces both the mRNA and protein levels of DENN/MADD (n = 3). C qPCR quantification of relative DENN/MADD expression in CD3 T cells from AML patients (n = 13) compared to (n = 5) HDs. (**p < 0.001) (D) (E) Correlation between DENN/MADD mRNA expression in T cells and miR-24-3p in EXOs and in T cells, quantified by qPCR. DENN/MADD expression in T cells is negatively correlated with (D) serum exosomal miR-24-3p level (R = –0.2460, p < 0.007) and (E) miR-24-3p in T cells ((R = –0.3, p < 0.007). The statistical analysis was performed with Spearman’s correlation and linear regression