PDF(Pubmed)
Abstract：
Blocking angiogenesis can inhibit tumor growth and metastasis. However, the mechanism underlying regulation of lung cancer angiogenesis remains unclear. The gap junction protein connexin 43 (Cx43) is implicated in angiogenesis. The aim of the present study was to determine the role of Cx43 in angiogenesis in vitro and its signaling pathways. Human pulmonary microvascular endothelial cells were transfected with Cx43-targeting siRNA or Cx43-overexpressing recombinant plasmid vector. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to determine Cx43, zonula occludens-1 (ZO-1), E-cadherin, β-catenin, von Willebrand factor (vWF), and plasminogen activator inhibitor-1 (PAI-1) mRNA and protein expression levels, respectively. Tyr265, Ser279, Ser368, and Ser373 phosphorylation levels in the C-terminus of Cx43 and intracellular and membranal Cx43 contents were determined using western blotting. Additionally, immunofluorescence, tube formation, Cell Counting Kit-8, and Transwell migration assays were performed. The results revealed that compared with that in the control samples, Cx43, ZO-1, E-cadherin, β-catenin, vWF, and PAI-1 mRNA and protein expression were significantly increased in the Cx43 overexpression group and significantly decreased in the Cx43-knockdown group. Moreover, the phosphorylation level of Ser279 as well as cell proliferation and migration rates were markedly increased in the Cx43 overexpression group, and tube formation revealed that the potential of angiogenesis was also increased. Conversely, in the Cx43-knockdown group, the phosphorylation level of Ser279 and cell proliferation and migration rates were reduced, and the potential of angiogenesis was greatly impaired. Under Cx43 overexpression, membranal Cx43 content was significantly increased, whereas under Cx43 knockdown, it was significantly reduced. Therefore, Cx43 overexpression could induce pulmonary angiogenesis in vitro by promoting cell proliferation and migration and activating ZO-1, E-cadherin, β-catenin, vWF, and PAI-1. This may be achieved by promoting phosphorylation and activation of the intracellular signal site Ser279 at the C-terminus of Cx43.
摘要：
阻断血管生成可以抑制肿瘤的生长和转移。然而,肺癌血管生成的潜在调节机制尚不清楚.间隙连接蛋白连接蛋白43(Cx43)与血管生成有关。本研究的目的是确定Cx43在体外血管生成及其信号通路中的作用。用靶向Cx43的siRNA或Cx43过表达的重组质粒载体转染人肺微血管内皮细胞。进行逆转录定量聚合酶链反应和蛋白质印迹以确定Cx43，小带闭塞-1（ZO-1），E-cadherin,β-连环蛋白，血管性血友病因子(vWF)，和纤溶酶原激活物抑制剂-1（PAI-1）mRNA和蛋白表达水平，分别。使用蛋白质印迹法测定Cx43C末端的Tyr265，Ser279，Ser368和Ser373磷酸化水平以及细胞内和膜Cx43含量。此外,免疫荧光，管形成,进行细胞计数试剂盒-8和Transwell迁移测定。结果表明,与对照样品相比,Cx43，ZO-1，E-钙粘蛋白，β-连环蛋白，vWF,在Cx43过表达组中，PAI-1mRNA和蛋白表达显着增加，而在Cx43敲低组中则显着降低。此外，在Cx43过表达组中，Ser279的磷酸化水平以及细胞增殖和迁移率均显著增加，和管的形成表明血管生成的潜力也增加。相反，在Cx43击倒组中，Ser279的磷酸化水平和细胞增殖和迁移率降低，血管生成的潜力大大受损。在Cx43过表达下，膜质Cx43含量显著增加，而在Cx43击倒下，它大大减少了。因此,Cx43过表达可通过促进细胞增殖和迁移以及激活ZO-1、E-cadherin,β-连环蛋白，vWF,和PAI-1。这可以通过促进Cx43的C末端的细胞内信号位点Ser279的磷酸化和活化来实现。
