MiR-10a-induced transcriptional inhibition of hoxd4 is related to promoter-associated ncRNAs. (a) Effects of promoter target site on siRNA-induced inhibition of hoxd4 expression in MCF7 cells. Si-nc34 and si-nc36 inhibited hoxd4 mRNA expression in MCF7 cells, whereas siD1 and siD2 had almost no effect in hoxd4 mRNA expression. (b) RT-PCR assay of ncRNAs in the hoxd4 promotor region of MCF7 cells after transfection with si-nc34, si-nc35 and si-nc36. Si-nc35 did not affect the expression level of nc-hoxd4-35. (c) Schematic representation of the siP1 and siP2 target sites on hsa-miR-10b. SiP1 and SiP2 were designed to target the stem and lopp region, respectively, of has-miR-10b precusor. (d) Hoxd4 expression after transfection of MCF7 cells with siP1 and siP2. SiP2 couldn't affect the hoxd4 mRNA expression. (e) Expression of hoxd4 and hoxb4 (relative to β-actin) in MCF7 and MDA-MB-231 cells after transfection with miR-10a and miR-10b duplexes. MiR-10a and -10b couldn't target their own primary transcript to modulate downstream gene expression. (f) Hoxd4 expression in MCF7 cells after RNAi against Argonautes and Dicer mRNAs. The results suggested that Dicer, AGO1 and AGO3 are required for the induction of transcriptional inhibition by miR-10a duplexes.