Pancreatic cancer (PC) is an aggressive and metastatic gastrointestinal tumor with a poor prognosis. Persistent activation of the TGF-β/Smad signaling induces PC cell (PCC) invasion and infiltration via epithelial-to-mesenchymal transition (EMT). Hedgehog signaling is a crucial pathway for the development of PC via the transcription factors Gli1/2/3. This study aimed to investigate the underlying molecular mechanisms of action of hedgehog activation in TGF-β1-triggered EMT in PCCs (PANC-1 and BxPc-3). In addition, overexpression and shRNA techniques were used to evaluate the role of Smad4 in TGF-β1-treated PCCs. Our data showed that TGF-β1 promoted PCC invasion and infiltration via Smad2/3-dependent EMT. Hedgehog-Gli signaling axis in PCCs was activated upon TGF-β1 stimulation. Inhibition of hedgehog with cyclopamine effectively antagonized TGF-β1-induced EMT, thereby suggesting that the hedgehog signaling may act as a downstream cascade signaling of TGF-β1. As a key protein that assists the nuclear translocation of Smad2/3, Smad4 was highly expressed in PANC-1 cells, but not in BxPc-3 cells. Conversely, Gli1 expression was low in PANC-1 cells, but high in BxPc-3 cells. Furthermore, knockdown of Smad4 in PANC-1 cells by shRNA inhibited TGF-β1-mediated EMT and collagen deposition. Overexpression of Smad4 did not affect TGF-β1-mediated EMT due to the lack of significant increase in nuclear expression of Smad4. Importantly, Gli1 activity was upregulated by Smad4 knockdown in PANC-1 cells and downregulated by Smad4 overexpression in BxPc-3 cells, indicating that Gli1 may be a negative target protein downstream of Smad4. Thus, Smad4 regulates TGF-β1-mediated hedgehog activation to promote EMT in PCCs by suppressing Gli1 activity.

中文翻译：

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Smad4通过抑制Gli1活性调节TGF-β1介导的hedgehog激活以促进胰腺癌细胞的上皮间质转化

胰腺癌（PC）是一种侵袭性、转移性胃肠道肿瘤，预后较差。 TGF-β/Smad 信号传导的持续激活可通过上皮间质转化 (EMT) 诱导 PC 细胞 (PCC) 侵袭和浸润。 Hedgehog 信号传导是通过转录因子 Gli1/2/3 发展 PC 的重要途径。本研究旨在探讨 hedgehog 激活在 PCC（PANC-1 和 BxPc-3）中 TGF-β1 触发的 EMT 中作用的潜在分子机制。此外，使用过表达和shRNA技术来评估Smad4在TGF-β1处理的PCC中的作用。我们的数据表明，TGF-β1 通过 Smad2/3 依赖性 EMT 促进 PCC 侵袭和浸润。 PCC 中的 Hedgehog-Gli 信号轴在 TGF-β1 刺激下被激活。用环杷明抑制hedgehog可有效拮抗TGF-β1诱导的EMT，从而表明hedgehog信号传导可能作为TGF-β1的下游级联信号传导。 Smad4作为协助Smad2/3核转位的关键蛋白，在PANC-1细胞中高表达，但在BxPc-3细胞中不高表达。相反，Gli1 在 PANC-1 细胞中表达较低，但在 BxPc-3 细胞中表达较高。此外，通过shRNA敲低PANC-1细胞中的Smad4可抑制TGF-β1介导的EMT和胶原沉积。 Smad4 的过表达不影响 TGF-β1 介导的 EMT，因为 Smad4 的核表达没有显着增加。重要的是，PANC-1细胞中Smad4敲低可上调Gli1活性，BxPc-3细胞中Smad4过表达可下调Gli1活性，表明Gli1可能是Smad4下游的负靶蛋白。因此，Smad4 调节 TGF-β1 介导的 hedgehog 激活，通过抑制 Gli1 活性来促进 PCC 中的 EMT。