Institut de Génomique Fonctionnelle, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut National de la Santé et de la Recherche Médicale U661, and Université Montpellier 1 and 2, Montpellier F-34094, France.
Proc Natl Acad Sci U S A. 2010 Feb 9;107(6):2628-33. doi: 10.1073/pnas.0903747107. Epub 2010 Jan 25.
Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNA-mediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin down-regulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29DeltaSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekin-depleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.
Symplekin 是一种广泛表达的蛋白质，参与细胞质 RNA 的多聚腺苷酸化和转录调控，并且定位于上皮细胞的紧密连接（TJ）处。核 Symplekin 与 Y 框转录因子紧密连接相关的核核酸结合蛋白（ZONAB）合作，增加细胞周期相关基因的转录，同时抑制肠细胞的分化。我们在 12 个人结直肠癌（CRC）样本中的 8 个样本中检测到高水平的核 Symplekin。shRNA 介导的 Symplekin 表达减少足以显著降低 HT-29 CRC 细胞的无锚定依赖性生长和增殖，以及当注射到免疫缺陷动物中时它们的致瘤性。Symplekin 下调还被发现改变 TJ 中的离子转运，促进 ZONAB 在膜而不是核中的定位，并强烈增强 3D 基质中的细胞极化，导致围绕中央腔形成球体。Claudin-2 的表达在 Symplekin 下调后减少，当使用选择性 siRNA 下调 ZONAB 表达时，会出现类似的效果。病毒介导的 Claudin-2 表达恢复被发现可恢复 HT29DeltaSym 细胞中 ZONAB 的核表达，并挽救 Symplekin 下调对细胞极性、旁细胞转运、ZONAB 定位、细胞周期蛋白 D1 表达、增殖和无锚定依赖性生长诱导的表型改变。最后，siRNA 介导的 Claudin-2 下调增加了上皮细胞的跨上皮电阻，降低了细胞周期蛋白 D1 表达和 ZONAB 的核定位，与 Symplekin 耗尽细胞中的观察结果相似。我们的结果表明，核 Symplekin 的过度表达促进了人类结肠的肿瘤发生，而 Claudin-2 表达的调节是这种作用的关键。
