Research Paper Volume 10, Issue 12 pp 3662—3682

Figure 4. B3GALT5-AS1 bound to the promoter of miR-203 and repressed the expression of miR-203. (A) The subcellular distribution of B3GALT5-AS1 in the cytoplasmic and nuclear fractions of HCT116 cells was evaluated using cytoplasmic and nuclear RNA isolation followed by qRT-PCR.β-actin and U6 were used as cytoplasmic and nuclear controls, respectively. (B) Schematic outline of the predicted interaction sites between B3GALT5-AS1 and the promoter of miR-203. (C) The expression of miR-203 in B3GALT5-AS1 stably overexpressed and control HCT116 cells was detected using qRT-PCR. (D) The expression of miR-203 in B3GALT5-AS1 stably depleted and control SW620 cells was detected using qRT-PCR. (E) ChIRP assays in HCT116 cells were carried out with anti-sense probe sets specific for B3GALT5-AS1 or LacZ (negative control). The enriched DNA was measured using qRT-PCR with specific primers against miR-203 promoter. (F) Schematic outline of the constructed different depletion transcripts of B3GALT5-AS1. (G) After transient transfections of the different B3GALT5-AS1 expressing plasmids into HCT116 cells, miR-203 expression was measured using qRT-PCR. (H) After transient co-transfection of the firefly luciferase reporter containing the promoter of miR-203, renilla luciferase expression plasmid pRL-TK, and the different B3GALT5-AS1 expression plasmids into HCT116 cells, luciferase activities were detected using dual luciferase reporter assays. Results are displayed as the relative ratio of firefly luciferase activity to renilla luciferase activity. (I) After transient co-transfection of the firefly luciferase reporter containing the promoter of miR-203 and pRL-TK into B3GALT5-AS1 stably depleted and control SW620 cells, luciferase activities were measured by dual luciferase reporter assays. Results are shown as the relative ratio of firefly luciferase activity to renilla luciferase activity. (J) After transient transfections of the different B3GALT5-AS1 expressing plasmids into HCT116 cells, the expression of ZEB2 and SNAI2 was detected using qRT-PCR and western blot. (K) The expression of ZEB2 and SNAI2 in B3GALT5-AS1 stably depleted and control SW620 cells was detected using qRT-PCR and western blot. Data are displayed as mean ± s.d. of three independent experiments. **P < 0.01, ***P < 0.001, NS, not significant, Student’s t-test