microRNA miR-142-3p Inhibits Breast Cancer Cell Invasiveness by Synchronous Targeting of WASL, Integrin Alpha V, and Additional Cytoskeletal Elements
Fig 3
3’UTR luciferase assay and Western blotting identify Integrin alpha V and N-WASP as relevant regulatory targets of miR-142-3p.
(a) Alignment of the seed sequence of miR-142-3p with predicted target sites of the 3’UTR of ITGAV (top) and one of the three predicted target sites of WASL (bottom) according to the microRNA.org database [28]. (b,c) The 3’UTR of ITGAV is a target for transcriptional regulation by miR-142-3p. Cells were transfected with plasmid pEZX-MT01-ITGAV-3’UTR (b, upper panel) expressing firefly luciferase (hLuc) under the control of an SV40 enhancer and the 3’UTR of human ITGAV, and renilla luciferase (hRLuc) under constitutive control of the cytomegalovirus (CMV) promoter. Cells were cotransfected with a control pre-miR or pre-miR-142-3p and simultaneously assayed for activity of both luciferases 72h after transfection. (b) pA = poly-A tail, pUCOri = origin of replication, Kan/NeomycinR = antibiotic resistance genes. (c) miR-142-3p transfection induced a significant decrease in ITGAV-specific normalized firefly luciferase activity in MCF-7 and MDA-MB-468, but not in MDA-MB-231 cells (N = 3, *P<0.05, ***P<0.001). (d) The 3’UTR of WASL is a target for transcriptional regulation by miR-142-3p Cells were transfected with plasmid pEZX-MT01-WASL-3’UTR (b, lower panel). Cells were cotransfected with a control pre-miR or pre-miR-142-3p and simultaneously assayed for activity of both luciferases 72h after transfection. WASL-dependent luciferase activity is reduced ranging from 64% to 88% in all three cell lines (N = 3, ***P<0.001). (e-g) Western blot confirmation of differentially regulated Integrin-αV and N-WASP protein expression in miR-142-3p vs. control transfected breast cancer cell lines. miR-142-3p upregulation inhibits N-WASP expression in all investigated cell lines (e-g), which can be reversed by anti-miR inhibition. miR-142-3p precursor transfection inhibits Integrin-αV expression in MDA-MB-468 (f) and MCF-7 (g) cells. The migration position of molecular weight markers relative to Integrin-αV and N-WASP is shown in S4 Fig.