LAUSR

The regulation of tumor suppressor genes by microRNAs (miRNAs) is often demonstrated as a one-miRNA-to-one-target relationship. However, given the large number of miRNA sites within a 3′ untranslated region (UTR), most targets likely undergo miRNA cooperation or combinatorial action. Programmed cell death 4 (PDCD4), an important tumor suppressor, prevents neoplastic events and is commonly downregulated in cancer. ABSTRACT The regulation of tumor suppressor genes by microRNAs (miRNAs) is often demonstrated as a one-miRNA-to-one-target relationship. However, given the large number of miRNA sites within a 3′ untranslated region (UTR), most targets likely undergo miRNA cooperation or combinatorial action. Programmed cell death 4 (PDCD4), an important tumor suppressor, prevents neoplastic events and is commonly downregulated in cancer. This study investigates the relationship between miRNA 21 (miR-21) and miR-499 in regulating PDCD4. This was explored using miRNA overexpression, mutational analysis of the PDCD4 3′ UTR to assess regulation at each miRNA site, and 50% inhibitory concentration (IC50) calculations for combinatorial behavior. We demonstrate that the first miR-499 binding site within PDCD4 is inactive, but the two remaining sites are both required for PDCD4 suppression. Additionally, the binding of miR-21 to PDCD4 influenced miR-499 activity through an increase in its silencing potency and stabilization of its mature form. Furthermore, adjoining miRNA sites more than 35 nucleotides (nt) apart could potentially regulate thousands of 3′ UTRs, similar to that observed between miR-21 and miR-499. The regulation of PDCD4 serves as a unique example of regulatory action by multiple miRNAs. This relationship was predicted to occur on thousands of targets and may represent a wider mode of miRNA regulation.