Figure 3.

MiR-30e inhibits AKT and ERK1/2 pathways via targeting IRS1. (a) Sequence of the miR-30e binding site within the human IRS1 3′-UTR and a schematic diagram of the reporter construct showing the entire IRS1 3′-UTR sequence and the mutant IRS1 3′-UTR sequence. The mutant nucleotides of the IRS1 3′-UTR are labeled in red. (b) Luciferase assay on MDA-MB-231 cells, which were co-transfected with miR-NC or miR-30e and a luciferase reporter containing the full length of IRS1 3′-UTR (WT) or a mutant (MT) harboring four mutant nucleotides of the miR-30e binding site. Luciferase activities were measured 24 h post-transfection. MiR-30e markedly suppressed luciferase activity in IRS1 3′-UTR (WT) reporter constructs. The data respresent means ± SEM. for separate transfections (n = 4). (c) The immunoblotting showed that expression levels of IRS1 were decreased in cells with miR-30e over-expression. (d) The expression levels of IRS1 in normal tissues and human BC specimens were determined by qRT-PCR analysis and fold changes were obtained by the ratios of IRS1 to GAPDH levels. (e) Spearman’s correlation analysis was used to determine the correlations between the expression levels of IRS1 and miR-30e in human BC specimens. (f) The expression levels of phosphorylated AKT (p-AKT) and phosphorylated ERK1/2 (p-ERK1/2) were decreased in cells with miR-30e over-expression, while AKT and ERK1/2 protein levels remained unchanged. Over-expression of IRS1 restored miR-30e-inhibited cellular protein levels of p-AKT and p-ERK1/2 and HIF-1α. (g) Overexpression of IRS1 rescued VEGF mRNA expression inhibited by miR-30e. The VEGF mRNA level was normalized to that of GAPDH. Data represent mean ± SD. of three replicates. **Indicates significant difference at P < 0.01. * or ^# indicated significant difference at P < 0.05. *Indicates significant difference compared to control; ^#indicates significant difference compared to miR-30e plus IRS1treatment.