Multiple microRNAs modulate p21Cip1/Waf1 expression by directly targeting its 3′ untranslated region

Cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as p21Cip1/Waf1, is a master downstream effector of tumor suppressors. In this study, we experimentally demonstrate through a high-throughput luciferase reporter screen that p21Cip1/Waf1 can be directly targeted by nearly 28 microRNAs (miRNAs). The results were further confirmed by a series of mutational analyses and luciferase reporter assays. These 28 miRNAs can substantially inhibit p21Cip1/Waf1 expression, predominantly at translational level. Many of these miRNAs were upregulated in cancers and might serve as modulators of oncogenesis. Furthermore, 8 of these 28 p21-regulating miRNAs are located in the chromosome 19 miRNA cluster, the largest miRNA gene cluster in humans, and they can clearly promote cell proliferation and cell-cycle progression in choriocarcinoma cells. In conclusion, our screening strategy provides an alternative approach to uncovering miRNA modulators of an individual mRNA, and it has identified multiple miRNAs that can suppress p21Cip1/Waf1 expression by directly targeting its 3′ untranslated region.