Departemt of Geriatric Surgery, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China.
Cancer Cell Int. 2013 Feb 21;13(1):18. doi: 10.1186/1475-2867-13-18.
The loss of tumor suppressor gene expression is involved in the carcinogenesis of gastric cancer (GC). Klotho is a recently identified tumor suppressor gene that epigenetically inactivated in gastric cancer. However, the signaling pathways involved in the suppressive role of klotho have rarely been reported in gastric cancer. In this study, we investigated the involvement of klotho in gastric cancer cell proliferation, apoptosis, and autophagy as well as the associated signaling.
Methylation of klotho gene promoter in GC-7901, MNK-45 and AGS gastric cancer cells as well as GES-1 normal gastric epithelial cells was detected by bisulfate-based PCR. Restoration of klotho gene expression was established by applying a demethylating agent and delivering aklotho gene expression vector into GC-7901 cells. Cell viability was measured by CCK-8 assay. Cell apoptosis and cycling were analyzed by flow cytometry. Autophagy was measured by detecting LC3-I and LC3-II expression. Protein levels and phosphorylation were measured by Western blot assay.
Methylation of klotho gene promoter and expression of the klotho gene were detected in GC cells. Restoration of klotho gene expression significantly inhibited cell proliferation, induced cell apoptosis, and increased LC3-I/LC3-II expression in GC cells. Restoration of klotho gene expression downregulated the phosphorylation levels of IGF-1 receptor, IRS-1, PI3K, Akt, and mTOR proteins. Both apoptosis and autophagy inhibitors blocked klotho-induced apoptosis and autophagy.
Klotho is a tumor suppressor in gastric cancer, which regulates IGF-1R phosphorylation and the subsequent activation of IRS-1/PI3K/Akt/mTOR signaling, tumor cell proliferation, apoptosis, and autophagy.
肿瘤抑制基因表达的丧失与胃癌（GC）的发生有关。Klotho 是最近发现的一种肿瘤抑制基因，在胃癌中被表观遗传失活。然而，Klotho 在胃癌中的抑制作用的信号通路很少有报道。在这项研究中，我们研究了 Klotho 如何参与胃癌细胞的增殖、凋亡和自噬以及相关的信号通路。
通过亚硫酸氢盐基 PCR 检测 GC-7901、MNK-45 和 AGS 胃癌细胞以及 GES-1 正常胃上皮细胞中 Klotho 基因启动子的甲基化。通过应用去甲基化剂和将 klotho 基因表达载体转染到 GC-7901 细胞中来建立 Klotho 基因表达的恢复。通过 CCK-8 测定法测量细胞活力。通过流式细胞术分析细胞凋亡和细胞周期。通过检测 LC3-I 和 LC3-II 的表达来测量自噬。通过 Western blot 测定法测量蛋白质水平和磷酸化。
在 GC 细胞中检测到 Klotho 基因启动子的甲基化和 Klotho 基因的表达。Klotho 基因表达的恢复显著抑制 GC 细胞的增殖，诱导细胞凋亡，并增加 GC 细胞中的 LC3-I/LC3-II 表达。Klotho 基因表达的恢复下调了 IGF-1 受体、IRS-1、PI3K、Akt 和 mTOR 蛋白的磷酸化水平。凋亡和自噬抑制剂均阻断了 Klotho 诱导的凋亡和自噬。
Klotho 是胃癌中的一种肿瘤抑制因子，它调节 IGF-1R 磷酸化以及随后 IRS-1/PI3K/Akt/mTOR 信号通路的激活、肿瘤细胞的增殖、凋亡和自噬。
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