Figure 4

MiR-10a-induced transcriptional gene inhibition is associated with DNA methylation of the hoxd4 promoter. (a) Effect of 5-aza-dC treatment on the p15, gapdh and miR-10a expression levels in MCF7 cells. Expression of p15 was upregulated when after treatment with 5-aza-dC, but no changes were observed in the expression level of miR-10a and gapdh gene. (b) Quantitative RT-PCR analysis of hoxd4 expression after 5-aza-dC treatment of MCF7 and MDA-MB-231 cells (c) 5-aza-dC reverses miR-10a inhibition of hoxd4 gene expression in MCF7 and MDA-MB-231 cells (□: Mock; ■: miR-10a: : Anti-miR-10a; : Mock + 5-aza-dC; □: miR-10a + 5-aza-dC; : Anti-miR-10a + 5-aza-dC). (d) Western blot analysis of H3K9me2 and H3K27me3 in the hoxd4 promotor. (e) ChIP analysis of H3K27me3 and H3K9me2 levels in two regions (box1 and box2) of the hoxd4 promotor (□: Cont; ■: miR-10a). (f) Bisulphate sequencing analysis of the hoxd4 promotor methylation status in MCF7 and MDA-MB-231 cells.