1. ¹Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY, USA
2. ²Center for Genetics and Molecular Medicine, School of Medicine, University of Louisville, Louisville, KY, USA
3. ³State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
4. ⁴Gene Regulation Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA

Correspondence: Dr Y Li, Department of Biochemistry and Molecular Biology, University of Louisville, 319 Abraham Flexner Way, Rm 513/A-building, Louisville, KY 40202, USA. E-mail: yong.li@louisville.edu

Received 23 August 2007; Revised 7 February 2008; Accepted 20 February 2008; Published online 31 March 2008.

Abstract

MicroRNAs (miRNAs) are small noncoding RNA molecules that negatively control expression of target genes in animals and plants. The microRNA-21 gene (mir-21) has been identified as the only miRNA commonly overexpressed in solid tumors of the lung, breast, stomach, prostate, colon, brain, head and neck, esophagus and pancreas. We initiated a screen to identify miR-21 target genes using a reporter assay and identified a potential miR-21 target in the 3'-UTR of the programmed cell death 4 (PDCD4) gene. We cloned the full-length 3'-UTR of human PDCD4 downstream of a reporter and found that mir-21 downregulated, whereas a modified antisense RNA to miR-21 upregulated reporter activity. Moreover, deletion of the putative miR-21-binding site (miRNA regulatory element, MRE) from the 3'-UTR of PDCD4, or mutations in the MRE abolished the ability of miR-21 to inhibit reporter activity, indicating that this MRE is a critical regulatory region. Western blotting showed that Pdcd4 protein levels were reduced by miR-21 in human and mouse cells, whereas quantitative real-time PCR revealed little difference at the mRNA level, suggesting translational regulation. Finally, overexpression of mir-21 in MCF-7 human breast cancer cells and mouse epidermal JB6 cells promoted soft agar colony formation by downregulating Pdcd4 protein levels. The demonstration that miR-21 promotes cell transformation supports the concept that mir-21 functions as an oncogene by a mechanism that involves translational repression of the tumor suppressor Pdcd4.