Figure 5.

miR-1293 Inhibited DNA Damage Repair by Downregulating BRD4 and DNA Repair Genes Synergistically In Vitro

(A–D) Evaluation of the effect of si-BRD4 and si-APEX1, si-RPA1, or si-POLD4. Western blot analysis (A, si-BRD4 and si-APEX1; B, si-BRD4 and si-RPA1; C, si-BRD4 and si-POLD4) and cell growth assay (D) in indicated cell lines after transfection with siRNA (si-NC [40 nmol/L]; si-BRD4, si-APEX1, si-RPA1, and si-POLD4 [each 20 nmol/L]). 120 h after transfection with the siRNAs, the cell growth rate was assessed with the CV staining assay using a relative ratio compared with that of si-NC-transfected cells. Error bar, SD for triplicate experiments. ∗p < 0.05. (E) Immunofluorescence analysis of HCT116^−/− cells that were double labeled with anti-γH2AX (red) and DAPI (blue; nuclei). Cells were transfected with 10 nmol/L miR-NC, miR-1293, si-NC, or the indicated siRNAs for 6 h following treatment with neocarzinostatin (200 ng/mL) for 30 min. (F) The percentage of ɣH2AX-positive cells in (E). ∗p < 0.05. (G) Homologous recombination (HR) repair analysis. U2OS DR-GFP cells were transfected with si-NC, the indicated siRNAs, miR-NC, or miR-1293, 6 h after transfection with the I-Sce1 endonuclease expression vector for 48 h. The HR efficiency of cells cotransfected with si-BRD4 and miR-1293 was compared with that of cells cotransfected with si-NC and miR-NC based on the percentage of GFP+ cells detected by flow cytometry. Error bar, SD for triplicate experiments. ∗p < 0.05. (H) Schematic models for the mechanism by which miR-1293 suppresses the DNA repair pathway and tumor growth.