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. 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 indicate they constitute sub-networks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho-ROCK signaling, invadopodia formation 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.

As well as providing insight to miR-200 function, this (and other Ago-HITS-CLIP experiments we have performed), provide further insights into general features of microRNA target site selection. Ongoing work suggests a wide variety of physical microRNA : mRNA interactions do not translate into functionally significant targeting, such as those occurring within the coding region or those mediated through imperfect seed-site interactions. Furthermore, only a limited number of more highly expressed microRNAs play significant gene targeting roles on a global scale, questioning the applicability of studies reliant upon exogenous microRNA over-expression.