Figure 2: miR-96 and miR-182 regulate the Palladin metastasis promoting protein.

(a) Stacked Venn diagram of the omic-data integration results. The outer circle represents the number of 'breast cancer genes' in PubMed (see Methods). The 2nd inner circle represents the number of genes with a conserved miRNA target site in their 3′-UTR based on TargetScan. The 3rd inner circle represents the number of genes with a known SNP in their miRNA target site based on dbSNP138common. The most inner circle represents the number of breast cancer genes with potential functional variants classified as 'cytoskeleton organization' by Gene Ontology. (b) Predicted binding sites for hsa-miR-96/182 on the PALLD 3′-UTR. rs1071738 PALLD SNP is marked in red. Three Luciferase constructs under regulation of the PALLD 3′-UTR were used for transient reporter assay experiments: negative control 3′-UTR (Target-deletion), G allele, and C allele. Below, sequences of mature hsa-miR-96 and hsa-miR-182 aligned to the target site with the ‘seed' regions marked in bold. (c) Luciferase activity 48 h following co-transfection with hsa-miR-96 or hsa-miR-182 in combination with either of the PALLD 3′-UTR constructs. (d) Palladin mRNA downregulation 24 h following overexpression of hsa-miR-96, hsa-miR-182 or pcDNA3 control plasmid in Hs578 cells. (e) Palladin protein downregulation (isoforms 3 and 4) 24 h following overexpression of hsa-miR-96, hsa-miR-182 or pcDNA3 control plasmid. (f) Palladin mRNA upregulation 24 h following downregulation of miR-96 or miR-182 (by antago-miRs or scrambled control) in MCF-7 cells. (g) Palladin protein downregulation (isoform 4) 2–3 weeks following stable overexpression of mmu-miR-96, mmu-miR-182 or scrambled control plasmid in 4T1 cells. (h) rs1071738 SNP genotype of T47D cell line (Sanger sequencing). Aligned below are four sequences: mature wild-type (WT) hsa-miR-96 and hsa-miR-182 (possessing a G nucleotide on the opposed position of the SNP), and mutant (MUT) miR-96 and miR-182, in which the G nucleotide was replaced by a C nucleotide. The seed regions (marked in bold) of the mutant miRNAs are fully complimentary to the binding site. (i) Palladin protein levels (isoform 3 and 4) 48 h following transfection of T47D cells by the indicated miRNAs (representative photos on the left). Values are presented as mean±s.e.m. (n=3, Student’s t-test *P<0.05, **P<0.01, ***P<0.005).