Figure 3.
Tumor suppressor role of miR-150-5p in PBL. (A) Spearman correlation between the expression of miR-150-5p and its targets BIRC5 and E2F3, as well as correlation between E2F3 and BIRC5 (E2F3 target). PBL and controls are represented in purple and gray, respectively. (B) Relative E2F3, BIRC5, and AURKB gene expression by reverse transcription polymerase chain reaction (RT-qPCR) at 72 hours. (C) E2F3, survivin, Aurora B, and p-Aurora B protein expression by western blot at 72 hours (left) and relative protein expression (right). (D) Relative luciferase activity (RQ) normalized to Renilla activity comparing HEK-293T cells transfected with miR-150-5p mimic or with NC miRNA and plasmid that contains luciferase gene followed by BIRC5 3′untranslated region. (E) Flow cytometry plots (left) of apoptosis at 24 hours by annexin V (Pacific Blue)/TO-PRO-3 (APC) staining and flow cytometry; plots (right) of relative viability and percentage of apoptotic cells (early plus late) of cells nucleofected with miRNA NC and miR-150-5p mimic, normalized to PBL-1 nontreated cells. (F) Cell cycle at 24 hours by flow cytometry and staining with TO-PRO-3 and flow cytometry. The percentage of cells in each phase of the cell cycle is shown. (G) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicates cell proliferation of cells treated with NC miRNA or miR-150-5p mimic at 0 hour and after 24, 48, and 72 hours of treatment. All figures represent PBL-1 nucleofected with NC miRNA in gray and with miR-150-5p mimic in blue. The t test was used for statistical analysis; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. All experiments were performed in triplicates unless otherwise specified. NC, negative control; RQ, relative quantity.

Tumor suppressor role of miR-150-5p in PBL. (A) Spearman correlation between the expression of miR-150-5p and its targets BIRC5 and E2F3, as well as correlation between E2F3 and BIRC5 (E2F3 target). PBL and controls are represented in purple and gray, respectively. (B) Relative E2F3, BIRC5, and AURKB gene expression by reverse transcription polymerase chain reaction (RT-qPCR) at 72 hours. (C) E2F3, survivin, Aurora B, and p-Aurora B protein expression by western blot at 72 hours (left) and relative protein expression (right). (D) Relative luciferase activity (RQ) normalized to Renilla activity comparing HEK-293T cells transfected with miR-150-5p mimic or with NC miRNA and plasmid that contains luciferase gene followed by BIRC5 3′untranslated region. (E) Flow cytometry plots (left) of apoptosis at 24 hours by annexin V (Pacific Blue)/TO-PRO-3 (APC) staining and flow cytometry; plots (right) of relative viability and percentage of apoptotic cells (early plus late) of cells nucleofected with miRNA NC and miR-150-5p mimic, normalized to PBL-1 nontreated cells. (F) Cell cycle at 24 hours by flow cytometry and staining with TO-PRO-3 and flow cytometry. The percentage of cells in each phase of the cell cycle is shown. (G) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicates cell proliferation of cells treated with NC miRNA or miR-150-5p mimic at 0 hour and after 24, 48, and 72 hours of treatment. All figures represent PBL-1 nucleofected with NC miRNA in gray and with miR-150-5p mimic in blue. The t test was used for statistical analysis; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. All experiments were performed in triplicates unless otherwise specified. NC, negative control; RQ, relative quantity.

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