Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.
Division of Hematology and Oncology, Case Western Reserve University and University Hospitals Seidman Cancer Center, Cleveland, OH, USA.
Mol Oncol. 2018 Dec;12(12):2124-2135. doi: 10.1002/1878-0261.12386. Epub 2018 Oct 23.
Protein inhibitor of activated STAT3 (PIAS3) is an endogenous suppressor of signal transducer and activator of transcription 3 (STAT3) signaling. By directly interacting with phosphorylated STAT3, PIAS3 can block the downstream transcriptional activity of STAT3, which is hyper-activated in various cancers. We previously reported that in malignant mesothelioma (MM), low PIAS3 expression is associated with increased STAT3 activation and correlates with poor patient survival, yet the regulatory mechanism(s) governing PIAS3 expression in MM remain unclear. Here, we demonstrate that PIAS3 protein expression does not correlate with its mRNA level in MM cell lines, indicating that PIAS3 expression is regulated at a post-transcriptional level. Inhibition of proteasomal degradation with MG132 (10 μm) or bortezomib (1 μm), alone and in combination, did not increase PIAS3 protein levels; furthermore, inhibition of protein synthesis by cycloheximide treatment did not decrease PIAS3 levels within 48 h, suggesting that PIAS3 expression is not actively regulated at a post-translational level. To determine whether miRNA (miRs) can translationally regulate PIAS3 expression, we combined miR microarray analysis with bioinformatic screening to identify candidate miRs, in MM cell lines with low PIAS3 expression, followed by luciferase reporter assays to validate miR regulation of the PIAS3 3'UTR. We identified miR-18a as a suppressor of PIAS3 expression that is upregulated in MM cells and whose inhibition can increase PIAS3 expression and suppress STAT3 activity. Moreover, we showed that miR-18a inhibition can decrease MM cell viability and that its expression is negatively correlated with MM patient survival. Taken together, these results suggest that targeting miR-18a may have therapeutic benefit in MM.
信号转导子和转录激活子 3（STAT3）的蛋白抑制剂（PIAS3）是 STAT3 信号的内源性抑制剂。PIAS3 通过与磷酸化的 STAT3 直接相互作用，可以阻断 STAT3 的下游转录活性，而 STAT3 在各种癌症中过度激活。我们之前报道过，在恶性间皮瘤（MM）中，低 PIAS3 表达与 STAT3 激活增加有关，并且与患者生存不良相关，但调控 MM 中 PIAS3 表达的调节机制尚不清楚。在这里，我们证明 PIAS3 蛋白表达与 MM 细胞系中的 mRNA 水平不相关，表明 PIAS3 表达受转录后水平调控。用 MG132（10 μM）或硼替佐米（1 μM）单独或联合抑制蛋白酶体降解，均不能增加 PIAS3 蛋白水平；此外，用环己酰亚胺处理抑制蛋白合成，在 48 小时内也不会降低 PIAS3 水平，表明 PIAS3 表达不是在翻译后水平上主动调控的。为了确定 miRNA（miRs）是否可以翻译调控 PIAS3 表达，我们结合 miR 微阵列分析和生物信息筛选，鉴定出在 PIAS3 低表达的 MM 细胞系中，具有候选 miR，然后进行荧光素酶报告基因检测，以验证 miR 对 PIAS3 3'UTR 的调控。我们发现 miR-18a 是 PIAS3 表达的抑制剂，在 MM 细胞中上调，其抑制可以增加 PIAS3 表达并抑制 STAT3 活性。此外，我们表明抑制 miR-18a 可以降低 MM 细胞活力，并且其表达与 MM 患者的生存呈负相关。总之，这些结果表明，针对 miR-18a 可能对 MM 具有治疗益处。
