Figure 5

p53-dependent regulation of miRNA (miR)-34b expression by 17β-estradiol (E2). (A) Reporter plasmids in which the luciferase coding sequence that had fused to the miR-34b promoter region were transfected into 293T cells in conjunction with either pcDNA3.1 plasmid coding with ER were cotransfected. Normalized luciferase activities are presented (n = 4). (B) miR-34b expression after E2 treatment in a subset of breast cancer cells (MCF-7, T47D and MDA-MB-435) with different estrogen receptor (ER) and p53 status (n = 3). The results are normalized to internal U6 snRNA expression and represent means ± SD. *P < 0.05. wt = wild type; mut = mutation. (C) Chromatin immunoprecipitation (ChIP) assays of MCF-7 cells treated with or without 10 nM E2. The results of PCRs of the two p53-responsive elements after ChIP with ERα or p53 in MCF-7 cells are indicated. Input = 1% of total lysate. IgG = immunoglobulin G. (D) Quantitative PCR of miR-34b expression in MCF-7 cells following 10 nM E2 treatment (24 hours) and 2 μM 5-aza-2'-deoxycytidine (5-AZA) (72 hours) (n = 3). The results are normalized to internal U6 snRNA expression and represent means ± SD. *P < 0.05. CTRL = control. (E) Methylation-specific PCRs of miR-34b/c CpG islands in MCF-7 cells after 10 nM E2 treatment. Bands in the ''M'' lanes are PCR products obtained with methylation-specific primers. Those in the ''U'' lanes are products obtained with non-methylation-specific primers. (F) Bisulfite sequencing of miR-34b/c CpG islands in the indicated breast cancer cell lines with the same treatments shown in part (D). Open and filled circles represent unmethylated and methylated CpG sites, respectively. Each treatment group was assayed in seven replicate experiments.