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Keywords:

  • cytoskeleton;
  • HITS-CLIP;
  • invadopodia;
  • microRNA;
  • miR-200

Abstract

The microRNAs of the miR-200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago-HITS-CLIP technology for transcriptome-wide identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR-200a and miR-200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR-200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR-200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR-200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.

Synopsis

Thumbnail image of graphical abstract

miR-200 microRNAs are involved in the maintenance of epithelial integrity. Direct, transcriptome-wide target detection and validation identifies genes functionally grouped as regulators of Rho GTPase signaling, invadopodia formation, metalloprotease activity and cell adhesion, which together regulate cell motility, migration and cancer metastasis.

  • The global profile of miR-200 targets in breast cancer cells reveals a network of cytoskeletal regulators
  • miR-200 is found to control invadopodia, focal adhesions and Rho GTPase signaling.
  • Canonical seed-3′UTR target site interactions are dominant, but target sites in coding regions and non-canonical interactions are also detected.
  • Target genes identified in cell lines negatively correlate with miR-200 across human breast cancer samples as well.