目的: 研究卡格列净（canagliflozin,GANA）抑制半乳糖凝集素-3（galectin-3,Gal-3）介导的肝内胆管癌细胞增殖与迁移的作用。方法: 采用TIMER2.0、GEPIA2及R2数据库分析Gal-3在胆管癌中的表达情况及其对胆管癌患者的总体生存率是否有影响;用免疫组化检测肝内胆管癌组织和癌旁组织中 Gal-3 的表达情况;利用分子对接分析卡格列净与Gal-3的结合情况;采用不同浓度的卡格列净处理肝内胆管癌细胞系HUCCT1细胞24、48 h,通过CCK8检测卡格列净对细胞存活率的影响,以16 μM的卡格列净处理细胞24 h后,采用EdU荧光标记法、细胞划痕实验等分析卡格列净对细胞的增殖和迁移能力的影响。结果: 生信结果分析显示Gal-3在胆管癌中显著高表达,免疫组化结果表明,Gal-3在肝内胆管癌组织中的表达显著增高,且Gal-3的高表达与胆管癌患者的总体生存率显著相关;分子对接结果显示卡格列净可能结合在不同于Gal-3保守糖识别域的新位点;随着给予卡格列净处理浓度的升高和处理时间的延长,细胞存活率降低,用16 μM卡格列净处理HUCCT1细胞24 h后,处理组的增殖能力显著低于对照组,处理组的细胞迁移速度明显低于对照组。结论: 卡格列净可通过结合Gal-3抑制肝内胆管癌细胞的增殖和迁移。
Abstract
Objective To study the role of canagliflozin in inhibiting galectin-3-mediated proliferation and migration of intrahepatic cholangiocarcinoma cells. Methods TIMER2.0, GEPIA2 and R2 databases were used to analyze the expression of Gal-3 in cholangiocarcinoma and whether it had any effect on the overall survival rate of cholangiocarcinoma patients. The expression of Gal-3 in intrahepatic cholangiocarcinoma tissues and paracarcinoma tissues was detected by immunohistochemistry. Molecular docking was used to analyze the binding of canagliflozin to Gal-3. Cell experiments were performed with the intrahepatic cholangiocarcinoma cell line HUCCT1, and cells were treated with different concentrations of canagliflozin for 24h and 48h, and the effect of canagliflozin on cell survival rate was detected by CCK8. After treating cells with 16μM canagliflozin for 24h, the effect of canagliflozin on the proliferation and migration of cells was analyzed by using EdU Fluorescent Labeling and the Wound-Healing Assay. Results Bioinformatics Analysis results showed that Gal-3 was significantly highly expressed in cholangiocarcinoma, and the immunohistochemical results using intrahepatic cholangiocarcinoma tissues and paracarcinoma tissues also showed that the expression of Gal-3 was significantly increased in intrahepatic cholangiocarcinoma, and that the high expression of Gal-3 was significantly correlated with the overall survival rate of the cholangiocarcinoma patients. The results of the molecular docking study revealed that canagliflozin is capable of recognizing Gal-3 at a novel site instead of the conventional carbohydrate binding site. As the concentration of canagliflozin treatment increased and the treatment time was prolonged, the cell survival rate decreased. After treating HUCCT1 cells with 16μM canagliflozin for 24 h, the results of EdU Fluorescent Labeling showed that the proliferative ability of the treated group was significantly lower than that of the control group. The results of the Wound-Healing Assay showed that the cell migration rate of the treated group was significantly lower than that of the control group. Conclusion Canagliflozin inhibits the proliferation and migration of intrahepatic cholangiocarcinoma cells by binding to Gal-3.
关键词
卡格列净 /
肝内胆管癌 /
半乳糖凝集素-3
Key words
canagliflozin /
intrahepatic cholangiocarcinoma /
galectin-3
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基金
湖南师范大学医学院2023年度开放基金“PF-4708671靶向Galectin-1调控PTEN/STAT3信号通路抑制肝内胆管癌的增殖与迁移”（KF2022017）; 湖南师范大学2023年大型仪器测试开放基金资助项目“Galectin-1通过调控NF-kappaB信号通路促进肝内胆管癌进展的作用及机制研究”（23CSY170）
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