ARTICLE ABSTRACTIntegrins are adhesion receptors involved in bidirectional signaling that are crucial for various cellular responses during normal homeostasis and pathologic conditions such as cancer progression and metastasis. Aberrant expression of noncoding microRNAs (miRNA) has been implicated in the deregulation of integrin expression and activity, leading to the development and progression of cancer tumors, including their acquisition of the metastatic phenotype. miR-31 is a key regulator of several critical genes involved in the invasion-metastasis cascade in cancer. Using diverse cell-based, genetic, biochemical, flow cytometry, and functional analyses, we report that miR-31 is a master regulator of integrins as it targets multiple α subunit partners (α2, α5, and αV) of β1 integrins and also β3 integrins. We found that expression of miR-31 in cancer cells resulted in a significant repression of these integrin subunits both at the mRNA and protein levels. Loss of expression of α2, α5, αV, and β3 was a direct consequence of miR-31 targeting conserved seed sequences in the 3′ untranslated region of these integrin subunits leading to their posttranscriptional repression, which was reflected in their diminished surface expression in live cells. The biological consequence of decreased the cell surface of these integrins was a significant inhibition of cell spreading in a ligand-dependent manner. Although different reports have shown that a single integrin can be regulated by several miRNAs, here we show that a single miRNA, miR-31, is able to specifically target several integrin subunits to regulate key aspects of cancer cell invasion and metastasis. Mol Cancer Res; 9(11); 1500–8. ©2011 AACR.