Figure 4.

Bcl-w Is a Direct Target Gene of miR-205-5p, Which Is Downregulated by IR

(A) Left, Venn diagram illustrating selected miR-205-5p, which is downregulated by IR and enhances the survival of patients as well as targets Bcl-w. Right, the probability of patient survival for miR-205-5p, which fulfilled above conditions, was searched using Kaplan-Meier analysis. Kaplan-Meier survival curves of miR-205-5p expression in breast cancer patients were based on METABRIC dataset. (B and C) Validation by western blot analysis and qRT-PCR of Bcl-w protein (B) and mRNA (C) expression after transfection of miR-205-5p in H460 and MDA-MB-231 cells. (D) Left, structure of reporter constructs containing Bcl-w 3′ UTR downstream of the luciferase ORF. Right, luciferase assay of MDA-MB-231 cells, transfected with firefly luciferase constructs containing wild-type (WT) or mutant type (MT) target sites of Bcl-w, and negative control or miR-205-5p are shown. (E) After metastatic MDA-MB-231 cells (1 × 10⁶ cells/mouse) were injected into 8-week-old nude mice, IR (three times per 2.5 Gy) was exposed to mice, and then blood of mice was collected at 21 days. Levels of Bcl-w mRNA and miR-205-5p expression were measured in serum of mice by qRT-PCR (n = 4 animals per group). miRNA level was normalized with U6, and mRNA level was normalized with GAPDH. (F) After IR exposure, the biogenesis of miR-205-5p was examined by qRT-PCR in H460 and MDA-MB-231 cells. (G) Methylation-specific PCR (MSP) for methylation status of miR-205-5p CpG islands was conducted in H460 and MDA-MB-231 cells. All data are expressed as the mean ± SD of three experiments (*p < 0.05, **p < 0.005, ***p < 0.0005, Student’s t test).