Fig. 5

PTEN-AKT signaling mediated the miR-29a-induced breast cancer metastasis. A Procedure for miR-29a target gene prediction and pathway analysis. B Top 5 pathways from KEGG analysis of the 4,013 predicted target genes of miR-29a. C Sequence alignment between PTEN 3’UTR and miR-29a. Wild type (WT) or point mutated (MT) PTEN 3′UTR were cloned into pGL-3 Luciferase reporter vector. D Luciferase reporter assay demonstrated inhibition of 3′UTR of WT PTEN by miR-29a, but not for mutated PTEN. E miR-29a suppressed the expression of PTEN at both mRNA and protein levels in the mammary tumors derived from MCF-10A-Src. F Western blot indicated suppression of PTEN in MCF-7 cells by knocking down of ERα. G Western blot indicated suppression of PTEN and induction of p-AKT, p-p65 and β-catenin by miR-29a overexpression in both MCF-10A-Src and MDA-MB-231 cells. H miR-29a induced the expression of EMT markers fibronectin, vimentin and snail in MDA-MB-231 cells, which was attenuated by transfection with pcDNA3.1-Pten. I miR-29a induced cell invasion, which was attenuated by transfection with pcDNA3.1-Pten in in MDA-MB-231 cells. J, K Application of a AKT inhibitor GDC-0068 attenuated the miR-29a-induced EMT gene expression (J) and miR-29a-induced cell invasion (K) in MDA-MB-231 cells. Data are presented as the mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ns means non-significance