1. ¹Department of Experimental Surgery and Molecular Oncology of Solid Tumors, Medical Faculty Mannheim, University of Heidelberg, and DKFZ Heidelberg, Mannheim, Germany
2. ²Gene Regulation Section, Basic Research Laboratory, National Cancer Institute, Frederick, MD, USA
3. ³Department of Surgery, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany

Correspondence: Professor H Allgayer, Department of Experimental Surgery/Molecular Oncology of Solid Tumors (Collaboration Unit German Cancer Research Center-DKFZ-Heidelberg), Medical Faculty Mannheim, Ruprecht-Karls-University Heidelberg, Mannheim 68167, Germany. E-mail: heike.allgayer@chir.ma.uni-heidelberg.de

Received 26 March 2007; Revised 14 September 2007; Accepted 21 September 2007; Published online 29 October 2007.

Abstract

Tumor-suppressor Pdcd4 inhibits transformation and invasion and is downregulated in cancers. So far, it has not been studied as to whether miRNAs, suppressing target expression by binding to the 3'-UTR, regulate Pdcd4 or invasion. The present study was conducted to investigate the regulation of Pdcd4, and invasion/intra-vasation, by miRNAs. A bioinformatics search revealed a conserved target-site for miR-21 within the Pdcd4-3'-UTR at 228–249 nt. In 10 colorectal cell lines, an inverse correlation of miR-21 and Pdcd4-protein was observed. Transfection of Colo206f-cells with miR-21 significantly suppressed a luciferase-reporter containing the Pdcd4-3'-UTR, whereas transfection of RKO with anti-miR-21 increased activity of this construct. This was abolished when a construct mutated at the miR-21/nt228–249 target site was used instead. Anti-miR-21-transfected RKO cells showed an increase of Pdcd4-protein and reduced invasion. Moreover, these cells showed reduced intra-vasation and lung metastasis in a chicken–embryo–metastasis assay. In contrast, overexpression of miR-21 in Colo206f significantly reduced Pdcd4-protein amounts and increased invasion, while Pdcd4-mRNA was unaltered. Resected normal/tumor tissues of 22 colorectal cancer patients demonstrated an inverse correlation between miR-21 and Pdcd4-protein. This is the first study to show that Pdcd4 is negatively regulated by miR-21. Furthermore, it is the first report to demonstrate that miR-21 induces invasion/intravasation/metastasis.