MicroRNA-224 Induces G1/S Checkpoint Release in Liver Cancer

Profound changes in microRNA (miR) expression levels are frequently found in liver cancers compared to the normal liver. In this study, we evaluate the expression of miR-224 in human HCC and CCA, as well as its downstream targets and affected pathways. We show that miR-224 is upregulated in a large cohort of human CCA, similar to its upregulation in human HCC. For the purpose of studying the roles of miR-224 in HCC and CCA, we enforced miR-224 expression in cells. mRNA arrays followed by Ingenuity Pathway Analysis (IPA)-identified putative molecules and pathways downstream of miR-224. Phenotypically, we report that enforced expression of miR-224 increases the growth rate of normal cholangiocytes, CCA cell lines, and HCC cell lines. In addition, we identified, in an unbiased fashion, that one of the major biologic processes affected by miR-224 is Gap1 (G1) to Synthesis (S) transition checkpoint release. We next identified p21, p15, and CCNE1 as downstream targets of miR-224 and confirmed the coordinated downregulation results in the increased phosphorylation of Retinoblastoma (Rb) with resulting G1/S checkpoint release. Our data suggest that miR-224 is a master regulator of cell cycle progression, and that its overexpression results in G1/S checkpoint release followed by accelerated cell growth.