Abstract
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.
Highlights
MicroRNAs are endogenous small non-coding RNAs comprised of approximately 19–25 nucleotides
This way, we wanted to reflect the heterogeneity of the disease, and investigate if the impact of miR-142-3p on breast cancer cell behaviour is uniform, or restricted to certain subtypes. qPCR analysis in the cell lines MDA-MD-231 (mesenchymal morphology, estrogen receptor negative (ER–), claudinlow, highly invasive and tumorigenic in nude mice), MDA-MB-468 and MCF-7, revealed about 80-fold higher miR-142-3p expression in MDA-MB-231 cells compared to MDA-MB-468 and MCF-7, which exhibited similar expression levels (S1 Fig)
The relatively higher expression of miR-142-3p in MDA-MB-231 cells compared to MCF-7 and MDA-MB-468 may be the reason for the efficiency of antimiR-inhibition in these cells, whereas MCF-7 and MD-MB-468 already displayed low miR-142-3p expression, and were less prone to the inhibitory approach
Summary
MicroRNAs (miRNAs) are endogenous small non-coding RNAs comprised of approximately 19–25 nucleotides. The functional role of miR-142-3p in breast cancer cells has not been elucidated, yet. Aberrant expression or function of miR-142-3p has been noted in several additional forms of cancer, including clear-cell kidney cancer, non-small cell lung cancer, hepatocellular carcinoma, colon cancer and thyroid cancer [15,16,17,18]. We demonstrate that upregulated miR-142-3p expression levels in a panel of human breast cancer cell lines leads to inhibition of Integrin-αV, a transmembrane receptor subunit implicated in matrix-dependent regulation of cell adhesion and motility [19,20,21]. Our data suggest a novel fundamental posttranscriptional mechanism of cell motility regulation by miR-142-3p, with translational implications for breast cancer pathogenesis and therapy
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