RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site

Figure 3

Dnd1 regulates Bim expression through Bim-3′UTR. (a) Constructs were prepared to express chimeric RNAs spanning the YFP with Bim-3′UTR or Bim-5′UTR as described in Section 2. (b and c) After MCF-7 cells were transfected with NC or Dnd1 siRNA for 24 h, the YFP-Bim-3′UTR (b) or YFP-Bim-5′UTR (c) vector was cotransfected for 24 h. These cells were then treated with actinomycin D (2.5 μg/mL) for the indicated times. YFP mRNA levels were measured by qRT-PCR and the percentage of YFP mRNA that remained was plotted. (d) After a 24-hour transfection with the YFP or YFP-Bim construct, the amounts of Dnd1 binding to the chimeric RNAs were analyzed by RIP assay followed by measurement of YFP mRNA levels by qRT-PCR. (e) qRT-PCR was used to measure the abundance of Bim mRNA present in the Dnd1-IP materials after the RIP assay was measured by qRT-PCR. (f) The mRNA level of Bim was examined in twenty-one pairs of breast tumors with adjacent mammary gland epithelial tissues via qRT-PCR analyses. (g) Correlation between the levels of Dnd1 and Bim. Values are mean ± SD. (, one-way ANOVA).

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