The Microtubule Network and Cell Death Are Regulated by an miR-34a/Stathmin 1/βIII-Tubulin Axis.
The Microtubule Network and Cell Death Are Regulated by an miR-34a/Stathmin 1/βIII-Tubulin Axis.
复制标题
DOI：
10.1158/1541-7786.mcr-16-0372
复制
发表时间：
2017-07
期刊：
影响因子：
--
通讯作者：
Sampson VB
中科院分区：
文献类型：
--
作者：
Vetter NS;Kolb EA;Mills CC;Sampson VB
microRNA-34 a（miR-34 a）是维持正常生理和疾病的信号网络的主要调节剂，目前正在开发作为基于miRNA的癌症治疗。先前的研究已经报道了骨肉瘤（OS）中miR-34 a的低表达;然而，OS中miR-34 a活性的分子机制尚未明确。因此，本研究评估了miR-34 a在调节影响OS中细胞死亡的信号转导途径中的作用。miR-34 a的水平在人OS细胞和儿科临床前测试联盟（PPTC）的异种移植物中减弱。生物信息学预测将stathmin 1（STMN 1）确定为潜在的miR-34 a靶标。生物素pulldown分析和荧光素酶报告基因分析证实了miR-34 a在STMN 1 mRNA 3′UTR内的靶向相互作用。在OS细胞中过表达miR-34 a抑制了STMN 1的表达并降低了体外细胞生长。miR-34 a的恢复导致微管不稳定和β III-微管蛋白表达增加，相应的G1/G2期细胞周期停滞和凋亡。通过siRNA沉默敲低Sp1转录因子也上调OS细胞中β III-微管蛋白的表达，表明miR-34 a间接影响Sp1。验证了miR-34 a在微管不稳定中的协调作用，当miR-34 a与微管抑制剂或化疗组合时，STMN 1磷酸化被抑制，并且在OS细胞中存在更大的细胞毒性。这些结果表明，miR-34 a直接抑制STMN 1基因和蛋白表达，上调β III-微管蛋白，导致微管网络破坏和细胞死亡。miR-34 a/STMN 1/β III-微管蛋白轴维持骨肉瘤中的微管细胞骨架，并且可以研究miR-34 a与微管抑制剂的组合作为新的治疗策略。
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 of miR-34a in microtubule destabilization, when miR-34a was combined with either microtubule inhibitors or chemotherapy, STMN1 phosphorylation was suppressed and there was greater cytotoxicity in OS cells. These results demonstrate that miR-34a directly represses STMN1 gene and protein expression and upregulates βIII-tubulin, leading to disruption of the microtubule network and cell death. The miR-34a/STMN1/βIII-tubulin axis maintains the microtubule cytoskeleton in osteosarcoma, and combining miR-34a with microtubule inhibitors can be investigated as a novel therapeutic strategy.