Zhang Jing, Zhang Xintong, Zhao Xiaoting, Jiang Mei, Gu Meng, Wang Ziyu, Yue Wentao
1 Department of Cellular and Molecular Biology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest Hospital, Capital Medical University, Beijing, China.
2 Central Laboratary, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, China.
Tumour Biol. 2017 Jul;39(7):1010428317703820. doi: 10.1177/1010428317703820.
Disregulation of dickkopf-related protein 1 (DKK1) has been reported in a variety of human cancers. However, how DKK1 functions in Non-small cell lung cancer has not been revealed. In the current study, DKK1 was knocked out by the lentivirus-mediated short hairpin RNA interference approach in H1299 and 95C non-small cell lung cancer cell lines. Subsequently, the migration and invasion ability were assessed by wound-healing and transwell assays. In addition, epithelial-mesenchymal transition markers and β-catenin were examined by Western blot analysis. The signaling pathway downstream of DKK1 was characterized using the Wnt signaling pathway inhibitor, IWP2, and glycogen synthase kinase 3 beta inhibitor, LiCl. Immunofluorescence analysis investigated the subcellular localization of β-catenin. The results suggested that knockdown of DKK1 caused reduced migration and invasion ability of H1299 and 95C cells. DKK1 silencing resulted in the downregulation of epithelial-mesenchymal transition-related proteins, such as Snail and zinc finger E-box binding homeobox 1. Besides, DKK1 silencing inhibited β-catenin and promoted the phosphorylation of β-catenin. Mechanism results indicated that the expression of β-catenin was reduced in H1299 or 95C cells after being treated with Wnt signaling inhibitor, IWP2. In addition, the inhibition of β-catenin phosphorylation by glycogen synthase kinase 3 beta inhibitor, LiCl, significantly enhanced the migration and invasion capacities in DKK1-knockdown cell lines. Furthermore, cell immunofluorescence revealed that nuclear β-catenin was reduced when DKK1 was knocked down. Taken together, these findings suggest that DKK1 induces the occurrence of epithelial-mesenchymal transition and promotes migration and invasion in non-small cell lung cancer cells. Mechanically, β-catenin plays a vital role in DKK1-induced non-small cell lung cancer cell migration and invasion, and DKK1 inhibits the phosphorylation of β-catenin, resulting in the increased nuclear localization of β-catenin.
已有报道称，在多种人类癌症中存在 dickkopf 相关蛋白 1（DKK1）失调的情况。然而，DKK1 在非小细胞肺癌中的作用机制尚未明确。在本研究中，通过慢病毒介导的短发夹 RNA 干扰方法，在 H1299 和 95C 非小细胞肺癌细胞系中敲除了 DKK1。随后，通过伤口愈合实验和 Transwell 实验评估细胞的迁移和侵袭能力。此外，采用蛋白质免疫印迹分析检测上皮-间质转化标志物和β-连环蛋白。利用 Wnt 信号通路抑制剂 IWP2 和糖原合酶激酶 3β抑制剂 LiCl 对 DKK1 下游的信号通路进行了表征。免疫荧光分析研究了β-连环蛋白的亚细胞定位。结果表明，敲低 DKK1 可导致 H1299 和 95C 细胞的迁移和侵袭能力降低。DKK1 沉默导致上皮-间质转化相关蛋白（如 Snail 和锌指 E 盒结合同源盒 1）表达下调。此外，DKK1 沉默抑制了β-连环蛋白，并促进了β-连环蛋白的磷酸化。机制研究结果表明，用 Wnt 信号抑制剂 IWP2 处理后，H1299 或 95C 细胞中β-连环蛋白的表达降低。此外，糖原合酶激酶 3β抑制剂 LiCl 抑制β-连环蛋白磷酸化，显著增强了 DKK1 敲低细胞系的迁移和侵袭能力。此外，细胞免疫荧光显示，敲低 DKK1 时，细胞核内的β-连环蛋白减少。综上所述，这些研究结果表明，DKK1 诱导上皮-间质转化的发生，并促进非小细胞肺癌细胞的迁移和侵袭。机制上，β-连环蛋白在 DKK1 诱导的非小细胞肺癌细胞迁移和侵袭中起关键作用，且 DKK1 抑制β-连环蛋白的磷酸化，导致β-连环蛋白核定位增加。
