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. 2019 Apr 15;10(5):332. doi: 10.1038/s41419-019-1565-6

Fig. 5. miR-142-3p decreases Bcl-2 expression and inhibits H3K27me3 demethylation by negatively regulating the target gene lysine demethylase 6A (KDM6A) in In vitro induced Tregs (iTreg) cells (n = 3).

Fig. 5

iTregs were expanded in vitro and treated or not treated with the inhibitor/mimic of miR-142-3p for 3 days. Following treatment, a KDM6A) inhibitor, GSK J4 (Selleckchem), was added to the control group and inhibitor group. a Representative example of Bcl-2 histogram for iTregs, with and without treatment. b Bcl-2 expression of iTregs in each group. c RNA was purified and quantitative RT-PCR (qRT-PCR) was used to determine the expression of Bcl-2 mRNA in each group. d qRT-PCR was used to determine Bcl-2 expression of the control group and inhibitor group treated or not treated with GSK J4. To determine whether human miR-142b-3p targets KDM6A mRNA, 293T cells were transduced with plasmids and luciferase reporters. e Schematic representation of the miR-142-3p target sequence within the 3′-UTR of KDM6A. Activity of the luciferase gene linked to the wild-type (WT) or mutant (MUT) 3′-UTR of KDM6A. Luciferase activities were measured after 48 h. The relative expression of luciferase in the miRNA-NC group was normalized to a value of 1. The results are presented as mean and standard deviation values for separate transfections (n = 3). KDM6A and H3K27me3 protein levels of iTregs, with and without treatment, determined using western blot (f and g, respectively). Results are presented as mean ± standard error of the mean values (*P < 0.05 and **P < 0.01)