Research Paper Volume 13, Issue 6 pp 8214—8227

Bromodomain-containing protein 4 silencing by microRNA-765 produces anti-ovarian cancer cell activity

miR-765-induced anti-ovarian cancer cell activity is due to BRD4 silencing. The lv-pre-miR-765-expressing pOC-1 cells (Stb-L1) were further transduced with or without an UTR-null BRD4 construct [BRD4 (UTR-null)], stable cell were established; cells with the scramble non-sense miRNA (lv-miRC) were utilized as control cells. Expression of BRD4 mRNA (A) and protein (B) as well as miR-765 (C) was shown; cells were further cultured for applied time periods, cell proliferation, migration and apoptosis were tested by EdU staining (D), “Transwell” (E) and TUNEL staining (F) assays, respectively, with results quantified (D–F). Stable pOC-1 cells expressing the lenti-CRISPR/Cas9-BRD4-KO construct, or the BRD4 KO cells, were further transduced with lv-pre-miR-765, miR-765 precursor anti-sense construct lentivirus (lv-antagomiR-765) or lv-miRC. Control cells were transfected with lenti-CRISPR/Cas9 control construct (“Cas9-C”), and stable cells established via selection by puromycin. Expression of BRD4 mRNA (G) and protein (H) as well as miR-765 (I) was shown; cells were further cultured for applied time periods, cell proliferation (J), migration (K) and apoptosis (L) were tested similarly. For each assay, n=5 (five replicate well/dishes). Data were presented as mean ± standard deviation (SD). #p A–F). * p G–L). Experiments in this figure were repeated five times with similar results obtained.

Figure 5. miR-765-induced anti-ovarian cancer cell activity is due to BRD4 silencing. The lv-pre-miR-765-expressing pOC-1 cells (Stb-L1) were further transduced with or without an UTR-null BRD4 construct [BRD4 (UTR-null)], stable cell were established; cells with the scramble non-sense miRNA (lv-miRC) were utilized as control cells. Expression of BRD4 mRNA (A) and protein (B) as well as miR-765 (C) was shown; cells were further cultured for applied time periods, cell proliferation, migration and apoptosis were tested by EdU staining (D), “Transwell” (E) and TUNEL staining (F) assays, respectively, with results quantified (DF). Stable pOC-1 cells expressing the lenti-CRISPR/Cas9-BRD4-KO construct, or the BRD4 KO cells, were further transduced with lv-pre-miR-765, miR-765 precursor anti-sense construct lentivirus (lv-antagomiR-765) or lv-miRC. Control cells were transfected with lenti-CRISPR/Cas9 control construct (“Cas9-C”), and stable cells established via selection by puromycin. Expression of BRD4 mRNA (G) and protein (H) as well as miR-765 (I) was shown; cells were further cultured for applied time periods, cell proliferation (J), migration (K) and apoptosis (L) were tested similarly. For each assay, n=5 (five replicate well/dishes). Data were presented as mean ± standard deviation (SD). #p < 0.05 (AF). * p < 0.05 vs. “Cas9-C” cells (GL). Experiments in this figure were repeated five times with similar results obtained.