National Institute of Cancer Research, National Health Research Institutes, Miaoli 35053, Taiwan.
Department of Otolaryngology, Head and Neck Collaborative Oncology Group, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan.
Neoplasia. 2020 Nov;22(11):554-565. doi: 10.1016/j.neo.2020.08.005. Epub 2020 Sep 19.
The cytokine-inducible Src homology 2-containing protein (CISH) is an endogenous suppressors of signal transduction and activator of transcription (STAT) and acts as a key negative regulator of inflammatory cytokine responses. Downregulation of CISH has been reported to associate with increased activation of STAT and enhanced inflammatory pathways. However, whether microRNAs (miRNAs) play a crucial role in CISH/STAT regulation in oral squamous cell carcinoma (OSCC) remains unknown. The expression of CISH on OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. Specific targeting by miRNAs was determined by software prediction, luciferase reporter assay, and correlation with target protein expression. The functions of miR-944 and CISH were accessed by transwell migration and invasion analyses using gain- and loss-of-function approaches. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR were used to evaluate the pro-inflammation cytokines expression under the miR-944, CISH, NNK or combinations treatment. We found that the CISH protein, which modulates STAT3 activity, as a direct target of miR-944. CISH protein was significantly down-regulated in OSCC patients and cell lines and its level was inversely correlated with miR-944 expression. The miR-944-induced STAT3 phosphorylation, pro-inflammation cytokines secretion, migration and invasion were abolished by CISH restoration, suggesting that the oncogenic activity of miR-944 is CISH dependent. Furthermore, tobacco extract (NNK) may contribute to miR-944 induction and STAT3 activation. Antagomir-mediated inactivation of miR-944 prevented the NNK-induced STAT3 phosphorylation and pro-inflammation cytokines secretion. Altogether, these data demonstrate that NNK-induced miR944 expression plays an important role in CISH/STAT3-mediated inflammatory response and activation of tumor malignancy.
细胞因子诱导的Src 同源 2 结构域包含蛋白（CISH）是信号转导和转录激活因子（STAT）的内源性抑制剂，作为炎症细胞因子反应的关键负调控因子。据报道，CISH 的下调与 STAT 的过度激活和炎症途径的增强有关。然而，miRNAs 是否在口腔鳞状细胞癌（OSCC）中的 CISH/STAT 调节中发挥关键作用仍不清楚。通过定量实时 PCR（qRT-PCR）和免疫组织化学测定了 CISH 在 OSCC 患者中的表达。通过软件预测、荧光素酶报告基因测定和与靶蛋白表达的相关性来确定 miRNA 的特异性靶向。通过转染迁移和侵袭分析，使用增益和失活方法评估 miR-944 和 CISH 的功能。酶联免疫吸附试验（ELISA）和 qRT-PCR 用于评估 miR-944、CISH、NNK 或组合处理下促炎细胞因子的表达。我们发现，CISH 蛋白作为 miR-944 的直接靶标，调节 STAT3 活性。CISH 蛋白在 OSCC 患者和细胞系中显著下调，其水平与 miR-944 表达呈负相关。CISH 恢复可消除 miR-944 诱导的 STAT3 磷酸化、促炎细胞因子分泌、迁移和侵袭，表明 miR-944 的致癌活性依赖于 CISH。此外，烟草提取物（NNK）可能有助于 miR-944 的诱导和 STAT3 的激活。反义寡核苷酸介导的 miR-944 失活可防止 NNK 诱导的 STAT3 磷酸化和促炎细胞因子分泌。总之，这些数据表明，NNK 诱导的 miR944 表达在 CISH/STAT3 介导的炎症反应和肿瘤恶性激活中起重要作用。
