microRNA miR-142-3p Inhibits Breast Cancer Cell Invasiveness by Synchronous Targeting of WASL, Integrin Alpha V, and Additional Cytoskeletal Elements

MicroRNAs (miRNAs, micro ribonucleic acids) are pivotal post-transcriptional regulators of gene expression. These endogenous small non-coding RNAs play significant roles in tumorigenesis and tumor progression. miR-142-3p expression is dysregulated in several breast cancer subtypes. We aimed at investigating the role of miR-142-3p in breast cancer cell invasiveness. Supported by transcriptomic Affymetrix array analysis and confirmatory investigations at the mRNA and protein level, we demonstrate that overexpression of miR-142-3p in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells leads to downregulation of WASL (Wiskott-Aldrich syndrome-like, protein: N-WASP), Integrin-αV, RAC1, and CFL2, molecules implicated in cytoskeletal regulation and cell motility. ROCK2, IL6ST, KLF4, PGRMC2 and ADCY9 were identified as additional targets in a subset of cell lines. Decreased Matrigel invasiveness was associated with the miR-142-3p-induced expression changes. Confocal immunofluorescence microscopy, nanoscale atomic force microscopy and digital holographic microscopy revealed a change in cell morphology as well as a reduced cell volume and size. A more cortical actin distribution and a loss of membrane protrusions were observed in cells overexpressing miR-142-3p. Luciferase activation assays confirmed direct miR-142-3p-dependent regulation of the 3’-untranslated region of ITGAV and WASL. siRNA-mediated depletion of ITGAV and WASL resulted in a significant reduction of cellular invasiveness, highlighting the contribution of these factors to the miRNA-dependent invasion phenotype. While knockdown of WASL significantly reduced the number of membrane protrusions compared to controls, knockdown of ITGAV resulted in a decreased cell volume, indicating differential contributions of these factors to the miR-142-3p-induced phenotype. Our data identify WASL, ITGAV and several additional cytoskeleton-associated molecules as novel invasion-promoting targets of miR-142-3p in breast cancer

Functional analysis of miR-142-3p upregulation on invasiveness, viability and size of MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells.

<p>Cells were transfected with a control miRNA, miR-142-3p or antimiR-142-3p as described in the methods section. (a,b) Matrigel invasion assay (BD Biosciences, Heidelberg, Germany) was performed as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143993#pone.0143993.ref009" target="_blank">9</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143993#pone.0143993.ref010" target="_blank">10</a>]. miR-142-3p significantly inhibited breast cancer cell invasiveness by 39% and 32% in MDA-MB-468 (24h) and MCF-7 cells (48h) respectively. In MDA-MB-231 cells, anti-miR-142-3p caused a significant increase in invasiveness of more than 50%. Error bars = s.e.m (n≥6, *P<0.05, ***P<0.01). (b) Right panel: representative micrographs of invasion filter membranes after crystal violet staining (MDA-MB-468). (c) MTT assay reveals no significant effect of miR-142-3p on cell viability. MDA-MB-231, MDA-MB-468 and MCF-7 cells transfected with miR-142-3p precursors or a control miR precursor were allowed to proliferate for 72h. Error bars = s.e.m., n≥3. (d-i) miR-142-3p upregulation reduces cancer cell diameter and volume. (d) The two-dimensional size [μm³] of transfected MDA-MB-231 and MDA-MB-468 cells revealed a significant decrease in size upon miR-142-3p upregulation. N>70, error bars = SEM, *P<0.05, ***P<0.01. (e-i) Digital holographic microscopy (DHM) of miRNA transfected MDA-MB-468 cells reveals decreased cell volume after miR-142-3p transfection. (e,g). Representative quantitative DHM phase contrast images of suspended transfected MDA-MB-468 cells (g) and control cells (e) (coded to 256 gray levels); the dashed circles illustrate the cell volume increase. (f, h) pseudo-three-dimensional representations of the phase images in e, g correspond to the projection of the cell thickness. (i) cell volume V of n = 251 miR-142-3p transfected cells appears significantly decreased in comparison to the volume of n = 172 control cells; Data are mean ± SEM, *P<0.05, ***P<0.001.</p

Nano-texture analysis.

<p>MDA-MB-468 cell surfaces without (left column) or with treatment by miR-142-3p (right column) were imaged by Atomic Force Microscopy (AFM) at nanometer resolution. Shown are (a) representative height profiles, (b) topography raw data of a (10 μm)² scan, (c) protruding structure elements (green), (d) the overlay of (a) and (b) in a 3D-map and (e) the number values for the object count and the total volume (given as the sum of individual sizes (LDVs, local deviational volumes) per image). Shown are the mean values ± SD per image for n = 5, ***P<0.001.</p

miR-142-3p induces a change in cellular morphology.

<p>MDA-MB-468 breast cancer cells were subjected to control precursor or miR-142-3p precursor transfection, and analyzed by phase contrast (a) and confocal immunofluorescence microscopy (b-e). Human skin fibroblasts were analysed for control purposes (c). Shown are phalloidin staining of actin filaments (red), staining of vinculin (red) and staining of Integrin-αV (green). Yellow colour denotes colocalization in merged images, whereas blue colour indicates DAPI nuclear staining in merged panels (d) and (e). (a) phase contrast image shows reduced size of miR-142-3p transfected cells compared to controls (scale bar = 200 μm). (b) miR-142-3p induces a change in cell morphology towards a more rounded shape with fewer membrane protrusions and less actin stress fibers. (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143993#pone.0143993.g004" target="_blank">Fig 4</a>). Confocal microscopy analyis of Z-stacks reveals localization of integrin-αV in a focal adhesion-like pattern in human skin fibroblasts (c), whereas a staining in cell-cell-contacts is observed in MDA-MB-468 cells (e). Shown are the top and bottom focal planes and projections. Use of secondary antibodies alone did not generate a signal in MDA-MB-468 cells (d). Compared to controls, miR-142-3p-transfected MDA-MB-468 cells showed a reduced accumulation of integrin-αV in cell-cell contacts (arrows). Scale bar in panels b-e = 10μm.</p

Expression of β-integrin subunits in human breast cancer cell lines.

<p>The expression of, αVβ3-, β1-, β3-, and β5- integrin in MDA-MB-468, MDA-MB-231 and MCF-7 cells was studied by confocal immunofluorescence (αVβ3) (a) or western blotting(β1-, β3-, β5-) (b), respectively, as described in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143993#pone.0143993.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143993#pone.0143993.g005" target="_blank">5</a>. Only MDA-MB-231 cells showed expression of (αV)β3-integrin, in partial colocalization with actin stress fibers in a fibroblast-like pattern. All breast cancer cell lines studied expressed β1-, and β5- integrin subunits (b), albeit in different quantities.</p

siRNA-mediated depletion of <i>WASL</i> or <i>ITGAV</i> reduces matrigel invasiveness of MCF-7 and MDA-MB-231 cells.

<p>(a) Confirmation of successful siRNA knockdown of <i>ITGAV</i> (left panel) and <i>WASL</i> (right panel) by qPCR. N = 4, ***P<0.001. (b) Confirmation of successful siRNA knockdown of integrin-αV and WASL at the protein level by Western blotting. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143993#pone.0143993.g003" target="_blank">Fig 3E–3G</a> for details. (c) ITGAV and WASL-depletion reduces matrigel invasiveness of MDA-MB-231 and MCF-7 cells. N>5, #P = 0.06 (n.s.), *P<0.05, ***P<0.001.</p

siRNA-mediated depletion of <i>WASL</i> or <i>ITGAV</i> differentially affect membrane protrusions, cell volume and dry mass of MDA-MB-468 cells.

<p>MDA-MB-468 cells were subjected to control, <i>ITGAV</i> or <i>WASL</i> siRNA treatment, followed by analysis via AFM (a) or DHM (b,c), 72h after transfection. (a) MDA-MB-468 cell surfaces were imaged by Atomic Force Microscopy (AFM) at nanometer resolution. Quantitative analysis of object counts as a readout of membrane protrusion formation reveals a significant reduction in cells treated with <i>WASL</i> siRNA (** = p<0.01, n = 15, error bars = s.e.m.). (b,c) Digital holographic microscopy of siRNA transfected MDA-MB-468 cells reveals decreased cell volume (b) and dry mass (c) after <i>ITGAV</i> siRNA transfection compared to control siRNA-transfected cells. *P<0.05, N = 200 cells per group, data are mean ± SEM.</p