Fig. 3: Characterization of AR Binding Sites on the miR-125b promoter and the correlation between AR and miR-125b. | Cell Death & Disease

Fig. 3: Characterization of AR Binding Sites on the miR-125b promoter and the correlation between AR and miR-125b.

From: Interrogation of gender disparity uncovers androgen receptor as the transcriptional activator for oncogenic miR-125b in gastric cancer

Fig. 3

A Schematics showing that the human 5′ UTRs of the miR-125b-1 and miR-125b-2 promoter region contain one (ARE1, -1303/-1308) and three (ARE2, -945/-950; ARE3, -754/-759; ARE4, -460/-465) AREs, respectively. B Putative human AREs identified by searching the promoter regions of miR-125b-1 and miR-125b-2 genes for a motif corresponding to the consensus sequence of ARE. A consensus sequence generated by WEBLOGO displays the blast results of four AREs locations in miR-125b-1 and miR-125b-2 promotors. C Electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts isolated from HGC-27 cells treated with DHT (50 nM) for 24 h. Digoxin-labeled probes span each of the four AR binding sites (ARE1-4) in the miR-125-1 and miR-125b-2 promoter. Deleted probes (Probe- ARE1-4 Del) were synthesized with a 6 bp deletion of the ARE on miR-125b-1/2 promoters. The specificity of the binding complex was also determined using the unlabeled wild probe as a competitor. The black, blue and red arrows indicate the specific binding complexes for each probe, respectively. D ChIP assays performed in HGC-27 and MGC-803 stably expressing FLAG-tagged AR cells treated ±DHT (50 nM) for 24 h. Immunoprecipitation was using the antibodies against AR (for HGC-27), Flag (for MGC-803), or IgG control. Input represents 10% of the total cell extract used for each immunoprecipitation. Lane M: 1 kb DNA marker (E) ChIP-qPCR for measuring AR binding sites (ARE1-4) at miR-125b-1/2 promoters in HGC-27 cells and MGC-803 transfected with AR-Flag vector after treated with ±DHT (50 nM) for 24 h. Specific PCR primers covering the ARE region of the miR-125b-1 or miR-125b-2 promoters were used for the PCR analysis, and data were presented as a ratio to the input. F Schematic diagram of the construction of luciferase reporter plasmid containing wild type (Wt) ARE and ARE deletion mutants (Del) used in this study. G 293-T cells were stimulated with DHT (50 nM) or vehicle control for 24 h after transfection with indicated ARE wild type or deletion plasmids together with a Renilla luciferase reporter gene and incubated for another 24 h. The luciferase activity was measured, and the firefly/renilla ratio was calculated for each dataset. Error bars are means ± SD of three independent experiments. (*P < 0.05, **P < 0.01) (H) The correlation between the expression level of AR and miR-125b in the tissues of 337 GC cases of the Tianjin cohort (left panel), 248 male cases (middle panel), and 89 female cases (right panel) measured by real-time RT-PCR (TaqMan). I The correlation between the expression level of AR and miR-125b in the tissues of 404 GC cases of TCGA GC cohort (left panel), 261 male cases middle panel), and 143 female cases (right panel).

Back to article page