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 associates with and silences BRD4 in ovarian cancer cells. MicroRNA-765 (miR-765) putatively binds to BRD4 3'-UTR (untranslated region) at position 801-810. (A) Subcellular expression (in cytosol and nuclear fractions) of endogenous miR-765 in pOC-1 primary ovarian cancer cells. (B) RNA pull-down assay results suggested directing binding between biotinylated-miR-765 and BRD4 mRNA in pOC-1 cells (C); the primary ovarian cancer cells (pOC-1 and pOC-2) (D–H) or the established cell lines (CaOV3 and SKOV3) (L, M) were transduced with the lentiviral construct encoding pre-miR-765 sequence (lv-pre-miR-765) or scramble non-sense miRNA (lv-miRC). After selection by puromycin stable cells were established, expression of miR-765 and listed genes was tested by qPCR and Western blotting assays (D, F–H, L, M), and results quantified; the BRD4 3’-UTR luciferase reporter activity was tested as well (E). The pOC-1 cells were transfected with the wild-type (WT) or the mutant miR-765 mimic (sequences listed in I), at 500 nM each for 36h, the relative BRD4 UTR luciferase reporter activity (J) and BRD4 mRNA expression (K) were shown. “Pare” stands for the parental control cells. “miRC” stands for microRNA control mimic. For each assay, n=5 (five replicate well/dishes). Data were presented as mean ± standard deviation (SD). * p

Figure 1. miR-765 associates with and silences BRD4 in ovarian cancer cells. MicroRNA-765 (miR-765) putatively binds to BRD4 3'-UTR (untranslated region) at position 801-810. (A) Subcellular expression (in cytosol and nuclear fractions) of endogenous miR-765 in pOC-1 primary ovarian cancer cells. (B) RNA pull-down assay results suggested directing binding between biotinylated-miR-765 and BRD4 mRNA in pOC-1 cells (C); the primary ovarian cancer cells (pOC-1 and pOC-2) (DH) or the established cell lines (CaOV3 and SKOV3) (L, M) were transduced with the lentiviral construct encoding pre-miR-765 sequence (lv-pre-miR-765) or scramble non-sense miRNA (lv-miRC). After selection by puromycin stable cells were established, expression of miR-765 and listed genes was tested by qPCR and Western blotting assays (D, FH, L, M), and results quantified; the BRD4 3’-UTR luciferase reporter activity was tested as well (E). The pOC-1 cells were transfected with the wild-type (WT) or the mutant miR-765 mimic (sequences listed in I), at 500 nM each for 36h, the relative BRD4 UTR luciferase reporter activity (J) and BRD4 mRNA expression (K) were shown. “Pare” stands for the parental control cells. “miRC” stands for microRNA control mimic. For each assay, n=5 (five replicate well/dishes). Data were presented as mean ± standard deviation (SD). * p < 0.05 vs. “lv-miRC”/“miRC” cells. Experiments in this figure were repeated five times with similar results obtained.