The Microtubule Network and Cell Death are Regulated by amiR-34a/Stathmin 1/βIII-tubulin Axis

摘要

MicroRNA-34a (miR-34a) is a master regulator of signaling networks that maintain normal physiology and disease and is currently in development as a miRNA-based therapy for cancer. Prior studies have reported low miR-34a expression in osteosarcoma (OS); however, the molecular mechanisms underlying miR-34a activity in OS are not well defined. Therefore, this study evaluated the role of miR-34a in regulating signal transduction pathways that influence cell death in OS. Levels of miR-34a were attenuated in human OS cells and xenografts of the Pediatric Preclinical Testing Consortium (PPTC). Bioinformatics predictions identified stathmin 1 (STMN1) as a potential miR-34a target. Biotin pulldown assay and luciferase reporter analysis confirmed miR-34a target interactions within the STMN1 mRNA 3′UTR. Overexpression of miR-34a in OS cells suppressed STMN1 expression and reduced cell growth in vitro. Restoration of miR-34a led to microtubule destabilization and increased βIII-tubulin expression, with corresponding G1/G2 phase cell cycle arrest and apoptosis. Knockdown of the Sp1 transcription factor, by siRNA silencing, also upregulated βIII-tubulin expression in OS cells, suggesting miR-34a indirectly affects Sp1. Validating the coordinating role ofmiR-34a in microtubule destabilization, when miR-34a was combined with eithermicrotubule inhibitors or chemotherapy, STMN1 phosphorylation was suppressed andthere was greater cytotoxicity in OS cells. These results demonstrate thatmiR-34a directly represses STMN1 gene and protein expression and upregulatesβIII-tubulin, leading to disruption of the microtubule network and celldeath.ImplicationsThe miR-34a/STMN1/βIII-tubulin axis maintains the microtubulecytoskeleton in osteosarcoma, and combining miR-34a with microtubuleinhibitors can be investigated as a novel therapeutic strategy.