Figure 2.

Aberrant expression of miR-545 modulates BRCA1 p220 expression and HRR of DSB. (A) DR-U2OS cells carrying a single copy of a stably integrated direct repeat of inactivated GFP genes (DR-GFP) repair a DSB generated at a defined I-SceI site, yielding a GFP signal as reported (Pierce et al. 1999). Asynchronously growing DR-U2OS cells were transiently transfected with a miR-545 mimic, a miR-545 antagomir (α-miR-545), or negative control reagents (NC-A and NC-B, respectively). Twenty-four hours later, they were transfected with the I-SceI restriction enzyme. Following 48 h of incubation, the number of GFP-positive cells was quantified by flow cytometry. (B) Western blot showing the expression of BRCA1 p220 in DR-U2OS cells 72 h after transfection with a miR-545 mimic, a miR-545 antagomir (α-miR-545), or negative controls. Actin served as a loading control. (C) Histograms reveal the relative enrichment of GFP-positive cells in miR-545 mimic- and miR-545 antagomir-transfected cells compared with respective controls. Overexpression of miR-545 resulted in significant suppression of HR efficiency, and inhibition of miR-545 led to an increased rate of HR-directed DSB repair. Data represent the average of the relevant assays results (n ≥ 3). Error bars indicate the SEM. P-values were determined by a Student's t-test; (*) P < 0.05; (***) P < 0.001.