Effect of targeting of syndecan-1 by microRNA miR-10b on breast cancer cell motility and invasiveness via a rho-GTPase- and E-cadherin-dependent mechanism.

Abstract

e21041 Background: microRNAs are small endogenous non-coding RNAs, which posttranscriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. Here, we investigatet the potential functional relationship of miR-10b and syndecan-1 in an in vitro study. Methods: MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behaviour was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. Results: miR-10b overexpression induced posttranscriptional downregulation of syndecan-1, as demonstrated by qPCR, flow cytometry, and 3’UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation were increased. Syndecan-1 co-immunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1 deficient cells revealed upregulation of ATF-2, COX- 2, cadherin-11, vinculin, ACTG2, MYL9, transgelin-1, RhoA/C, MMP2 and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, Cdk6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. Conclusions: We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.