MiR-10a modulates hoxd4 gene expression. (a) Quantitative PCR analysis of hoxd4 gene expression in MCF7 and MDA-MB-231 cells treated with miR-10a duplexes or mock oligos. (b) Protein analysis of Hoxd4 in MDA-MB-231 cells after transfection with miR-10a. When treated with 100 nM miR-10a duplexes, the expression of Hoxd4 protein was almost abolished compared to transfection with 50 nM miR-10a duplexes or control. (c) Effect of transfection of MCF7 cells with miR-10a duplexes on the relative luciferase activity of luciferase hoxd10 3'UTR and hoxd4 3'UTR reporter constructs. (d) Nuclear run-on assay of hoxd4 in the presence or absence of miR-10a and sid4. (e) RT-PCR analysis of hoxd4 expression in several human cells after transfection with miR-10a. The expression of hoxd4 mRNA was reduced after the transfection of miR-10a duplexes in all six cell types. (f) Schematic representation of the miR-10a-G5', -M and -G3' mutants. (g) Hoxd4 expression in MCF7 cells transfected with miR-10a and its mutants. The 3'most portion of miR-10a sequence is most important for miR-10a induced gene silence of hoxd4.