钙黏蛋白 17 对结直肠癌细胞增殖和凋亡的影响及其 PI3K/AKT/mTOR 信号通路调节机制. (Chinese)

Objective: To discuss the effect of Cadherin-17 on the proliferation and apoptosis of the colorectal cancer (CRC) cells, and to clarify its possible mechanism. Methods: The Cadherin-17 gene overexpression and small interference plasmids were constructed and packaged as the lentivirus and transfected into the SW480 cells to construct the stable transfection strain of over-expression and interference virus. The expression levels of Cadherin-17 mRNA and protein in the cells were detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western blotting methods, and the transfection efficiency was verified and the stable transfection strain was identified. The SW480 cells were divided into control group, empty vector group, Cadherin-17 over-expression plasmid (OV-Cadherin-17) group and Cadherin17 small interference plasmid (si-Cadherin-17) group. The activities of cells in various groups were detected by CCK-8 assay; the apoptotic rates of cells in various groups were detected by flow cytometry; the expression levels of Cadherin-17, B-cell lymphoma-2（Bcl-2), Bcl2-associated X (Bax), cytochrome c (Cyt-c) and cysteinyl aspartate specific proteinase-3（Caspase-3), and the phosphatidylinosital-3-kinase/ protein kinase B/mamalian target of repamycin (PI3K/AKT/mTOR) signaling pathway-related proteins in the cells in various groups were detected by Western blotting methods. The cells were treated with PI3K inhibitor LY294002 and divided into control group, LY294002 group, OV-Cadherin-17+LY294002 group, and si-Cadherin-17+LY294002 group; the proliferation activities and apoptotic rates of cells in various groups and the expression levels of Bcl-2, Bax, Cyt-c, Caspase-3 and the expression levels of PI3K/AKT/mTOR signaling pathway-related proteins in the cells in various groups were detected. Results: The RT-qPCR and Western blotting results showed that the OV-Cadherin-17 and si-Cadherin-17 transfection and stable transfection stain were successfully constructed. Compared with control group, the proliferation ability of the cells in OV-Cadherin-17 group was increased (P<0. 01), the apoptotic rate was decreased (P<0. 01), the expression levels of Bax and Caspase-3 proteins in the cells were decreased (P< 0. 01), the expression levels of Bcl-2 and Cyt-c proteins in the cells were increased (P<0. 01), and the expression levels of phosphorylated PI3K（p-PI3K), phosphorylated AKT（p-Akt), and phosphorylated mTOR（p-mTOR) proteins were increased (P<0. 01); the proliferation abilities of the cells in si-Cadherin17 and LY294002 groups were decreased (P<0. 01), the apoptotic rates were increased (P<0. 01), the expression levels of Bax and Caspase-3 proteins in the cells were increased (P<0. 01), the expression levels of Bcl-2 and Cyt-c proteins in the cells were decreased (P<0. 01), and the expression levels of p-PI3K, p-AKT, and p-mTOR proteins in the cells were decreased (P<0. 01); compared with LY294002 group, the proliferation ability of the cells in OV-Cadherin-17+LY294002 group was increased (P< 0. 01), the apoptotic rate was decreased (P<0. 01), the expression levels of Bax and Caspase-3 proteins in the cells were decreased (P<0. 01), the expression levels of Bcl-2 and Cyt-c proteins in the cells were increased (P<0. 01), the expression levels of p-PI3K, p-AKT, and p-mTOR proteins were increased (P<0. 01), the proliferation activity of the cells in si-Cadherin-17+LY294002 group was decreased (P< 0. 01), the apoptotic rate was increased (P<0. 01), the expression levels of Bax and Caspase-3 proteins in the cells were increased (P<0. 01), the expression levels of Bcl-2 and Cyt-c proteins in the cells were decreased (P<0. 01), and the expression levels of p-PI3K, p-AKT, and p-mTOR proteins were decreased (P<0. 01). Conclusion: Cadherin-17 can promote the proliferation and inhibit the apoptosis of the CRC cells, and its mechanism may be related to the activition of PI3K/AKT/mTOR signaling pathway regulated by Cadherin-17. [ABSTRACT FROM AUTHOR]

目的：探讨钙黏蛋白 17 (Cadherin-17) 对结直肠癌 (CRC) 细胞增殖和凋亡的影响，阐 明其可能的机制。方法：构建 Cadherin-17 基因过表达及小干扰质粒，包装成慢病毒，转染至 SW480 细胞，构建过表达和干扰病毒稳转株。采用实时荧光定量 PCR (RT-qPCR) 法和 Western blotting 法 检测细胞中 Cadherin-17 mRNA 和蛋白表达水平，验证转染效率并鉴定稳转株。SW480 细胞分为对照 组 、 空 载 体 组 、 Cadherin-17 过 表 达 质 粒 ( OV-Cadherin-17 ) 组 和 Cadherin-17 小 干 扰 质 粒 (si-Cadherin-17) 组，CCK-8 法检测各组细胞增殖活性，流式细胞术检测各组细胞凋亡率，Western blotting 法检测各组细胞中 Cadherin-17、B 细胞淋巴瘤 2 (Bcl-2)、Bcl-2相关X蛋白 (Bax) 、细胞色素c (Cyt-c)、含半胱氨酸的天冬氨酸蛋白水解酶 3 (Caspase-3) 和磷脂酰肌醇 3-激酶/蛋白激酶 B/哺乳动 物雷帕霉素靶蛋白 (PI3K/AKT/mTOR) 信号通路相关蛋白表达水平。采用 PI3K 抑制剂 LY294002 处理细胞，将细胞分为对照组、LY294002 组、OV-Cadherin-17+LY294002 组 和 si-Cadherin-17+ LY294002 组，检测各 组 细 胞 增 殖 活 性 、 细 胞 凋 亡 率 及 细 胞 中 Bcl-2、 Bax、 Cyt-c、 Caspase-3 和 PI3K/AKT/mTOR 信 号 通 路 相 关 蛋 白 表 达 水 平 。 结 果 ： RT-qPCR 和 Western blotting 法 检 测 ， OV-Cadherin-17 和 si-Cadherin-17 转染和稳转株构建成功。与对照组比较，OV-Cadherin-17 组细胞增殖 活性明显升高 (P<0. 01)，细胞凋亡率明显降低 (P<0. 01)，细胞中 Bax 和 Caspase-3 蛋白表达水平 明显降低 (P<0. 01)，Bcl-2 和 Cyt-c 蛋白表达水平明显升高 (P<0. 01)，磷酸化 PI3K (p-PI3K)、磷 酸化 AKT (p-AKT) 和磷酸化mTOR（p-mTOR) 蛋白表达水平明显升高 (P<0. 01)，si-Cadherin-17 组 和 LY294002 组细胞增殖活性明显降低 (P<0. 01)，细胞凋亡率明显升高 (P<0. 01)，细胞中 Bax 和 Caspase-3 蛋白表达水平明显升高 (P<0. 01)，Bcl-2 和 Cyt-c 蛋 白 表 达 水 平 明 显 降 低 (P<0. 01)， p-PI3K、p-AKT和p-mTOR蛋白表达水平明显降低 (P<0. 01)；与LY294002组比较，OV-Cadherin-17+ LY294002 组细胞增殖活性明显升高 (P<0. 01)，细胞凋亡率明显降低 (P<0. 01)，细胞中 Bax 和 Caspase-3 蛋白表达水平明显降低 (P<0. 01)，Bcl-2和Cyt-c蛋白表达水平明显升高（P<0. 01)，p-PI3K、 p-AKT 和 p-mTOR 蛋白表达水平明显升高 (P<0. 01)，si-Cadherin-17+LY294002 组细胞增殖活性明 显降低 (P<0. 01)，细胞凋亡率明显升高 (P<0. 01)，细胞中 Bax 和 Caspase-3 蛋白表达水平明显升 高 (P<0. 01)，Bcl-2 和 Cyt-c 蛋白表达水平明显降低 (P<0. 01)，p-PI3K、p-AKT 和 p-mTOR 蛋白 表达水平明显降低 (P<0. 01)。结论：Cadherin-17 可促进 CRC 细胞增殖，抑制 CRC 细胞凋亡，其机 制可能与 Cadherin-17 调控 PI3K/AKT/mTOR 信号通路的激活有关。 [ABSTRACT FROM AUTHOR]

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