Department of Occupational Health, College of Public Health, Zhengzhou University, Zhengzhou, 450001, China.
Mol Biol Rep. 2021 Mar;48(3):2365-2375. doi: 10.1007/s11033-021-06268-2. Epub 2021 Apr 1.
Epithelial-mesenchymal transition (EMT), transforming growth factor β(TGF-β) and reactive oxygen species(ROS) plays a central role in cancer metastasis. Moreover, nicotinamide adenine dinucleotide phosphate 4(NOX4) is one of the main sources of ROS in lung cancer cells suggesting that NOX4 is associated with tumor cell migration. NF-κB(Nuclear factor-Kappa-B) is known to regulate ROS-mediated EMT process by activating Snail transcription factor in A549 cells. The purpose of this study was to explore the relationship between NF-κB and NOX4 in ROS production during TGF-β induced EMT process. Several fractions have been pooled to evaluates the EMT process on lung cancer cells through real-time PCR, Western Blot and flow cytometry with DCFH-DA probe etc. Cells proliferation and migration activities were monitored by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and wound healing assay respectively. The result showed that TGF-β induction decreased the expression of E-cadherin, increased the Vimentin and the EMT transcription factor Snail in A549 cells. DPI (Diphenyleneiodonium chloride, an inhibitor of NOX4) inhibited the NOX4 expression and reduced ROS production induced by TGF-β, but didn't affect the activation of NF-κB induced by TGF-β (P > 0.05). BAY11-7082 (an inhibitor of NF-κB) inhibited the NF-κB (p65) expression and prevented the increase of NOX4 expression and ROS production induced by TGF-β (P < 0.001), which has also verified reduced TGF-β induced cell migration by inhibiting the EMT process, and also reduced cell proliferation of A549 cells (P < 0.001). The current research confirmed the TGF-β mediated EMT process via NF-κB/NOX4/ROS signaling pathway, NF-κB and NOX4 are likely to be the potential therapeutic targets for lung cancer metastasis.
上皮-间充质转化（EMT）、转化生长因子-β（TGF-β）和活性氧（ROS）在癌症转移中起着核心作用。此外，烟酰胺腺嘌呤二核苷酸磷酸 4（NOX4）是肺癌细胞中 ROS 的主要来源之一，表明 NOX4与肿瘤细胞迁移有关。NF-κB（核因子-Kappa-B）已知通过在 A549 细胞中激活 Snaill 转录因子来调节 ROS 介导的 EMT 过程。本研究旨在探讨 NF-κB 和 NOX4 在 TGF-β诱导的 EMT 过程中 ROS 产生中的关系。通过实时 PCR、Western Blot 和 DCFH-DA 探针等方法对肺癌细胞进行 EMT 过程评估，将几个馏分合并。细胞增殖和迁移活性分别通过 MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐）测定法和划痕愈合测定法进行监测。结果表明，TGF-β 诱导降低了 A549 细胞中 E-钙粘蛋白的表达，增加了波形蛋白和 EMT 转录因子 Snaill。DPI（二苯碘氯，NOX4 抑制剂）抑制了 TGF-β 诱导的 NOX4 表达和 ROS 产生，但不影响 TGF-β 诱导的 NF-κB 激活（P>0.05）。BAY11-7082（NF-κB 抑制剂）抑制了 NF-κB（p65）的表达，并阻止了 TGF-β 诱导的 NOX4 表达和 ROS 产生的增加（P<0.001），这也验证了通过抑制 EMT 过程减少了 TGF-β 诱导的细胞迁移，并且还减少了 A549 细胞的增殖（P<0.001）。目前的研究证实了 TGF-β 通过 NF-κB/NOX4/ROS 信号通路介导的 EMT 过程，NF-κB 和 NOX4 可能是肺癌转移的潜在治疗靶点。
