目的: 探讨半乳糖凝集素-1（Galectin-1,Gal-1）在二甲双胍抑制胆管癌细胞增殖中的作用及分子机制。方法: 以HuCCT1细胞为研究对象,给予不同剂量二甲双胍处理24、48、72 h,采用CCK8检测二甲双胍对细胞生存率的影响,以 0、20 mmol /L 的二甲双胍处理细胞 24 h,利用克隆形成实验、流式细胞仪、DAPI染色等分析二甲双胍对细胞克隆形成、细胞周期及细胞凋亡小体的影响;利用TCGA、GEPIA、UALCAN、HPA等数据库分析Gal-1在胆管癌中的表达及对胆管癌患者生存率的影响;免疫组化检测肝内胆管癌组织和癌旁组织中Gal-1的表达改变;以 0、20 mmol /L 的二甲双胍处理细胞 24 h,利用Western Blot技术检测Gal-1及与细胞增殖密切相关的蛋白PI3K、AKT、mTOR等的蛋白表达;进一步利用慢病毒转染技术构建Gal-1稳定沉默表达的HUCCT8细胞株,Western Blot分析Gal-1、PI3K、AKT、mTOR等的蛋白水平。结果: 随二甲双胍处理浓度的增高和处理时间的延长,细胞存活率显著降低,克隆形成实验结果显示,对照组和20 mmol /L二甲双胍处理组的克隆形成率分别为18.17% 和1.98%,二甲双胍处理组表现为显著的G2期细胞阻滞,且伴随细胞凋亡小体的大量形成。生信分析结果显示Gal-1在胆管癌细胞中表达显著增高,利用癌组织和癌旁组织进行免疫组化的结果与生信分析结果一致,且Gal-1的高表达与胆管癌患者的OS密切相关;二甲双胍处理的HuCCT1细胞中Gal-1的表达显著降低,并伴随PI3K/AKT信号通路的抑制;此外,抑制Gal-1的表达不仅可降低细胞的增殖能力还限制抑制PI3K/AKT信号通路。结论: 二甲双胍可通过抑制肝内胆管癌中Gal-1的表达,抑制PI3K/AKT信号通路的活化,进而导致细胞增殖抑制。
Abstract
Objective To investigate the role and molecular mechanism of galactose lectin-1(Galectin-1, Gal-1) in the inhibition of cholangiocarcinoma cell proliferation by metformin. Methods HuCCT1 cells were treated with different doses of metformin for 24, 48 and 72 h. The effect of metformin on cell survival was detected by CCK8, and cells were treated with 0 and 20 mmol /L of metformin for 24 h. The effects of metformin on cell clone formation, cell cycle and apoptotic vesicles were analyzed by clone formation assay, flow cytometry and DAPI staining. The expression of Gal-1 in cholangiocarcinoma and its effect on the survival rate of cholangiocarcinoma patients were analyzed using TCGA, GEPIA, UALCAN, HPA and other databases; the expression changes of Gal-1 in intrahepatic cholangiocarcinoma tissues and paracancerous tissues were detected by immunohistochemistry; cells were treated with 0 and 20 mmol /L metformin for 24 h, and Gal-1 and the protein PI3K, which is closely related to cell proliferation, were detected using Western Blot. The cells were treated with 0 and 20 mmol/L metformin for 24 h. The protein expression of Gal-1 and closely related proteins PI3K, AKT and mTOR were detected by Western Blot. Results The cell survival rate decreased significantly with the increase of metformin treatment concentration and treatment time, and the results of clone formation assay showed that the clone formation rates of control and 20 mmol/L metformin treated groups were 18.17% and 1.98%, respectively. The metformin treated groups showed significant G2 phase cell block and were accompanied by the formation of a large number of apoptotic vesicles. The results of biochemical analysis showed that Gal-1 expression was significantly increased in cholangiocarcinoma cells, and the results of immunohistochemistry using cancer and paraneoplastic tissues were consistent with the results of biochemical analysis, and the high expression of Gal-1 was closely associated with OS in cholangiocarcinoma patients; the expression of Gal-1 was significantly decreased in metformin-treated HuCCT1 cells, and accompanied by the inhibition of PI3K/AKT signaling pathway; in addition , inhibition of Gal-1 expression not only reduced cell proliferation ability but also restricted the inhibition of PI3K/AKT signaling pathway. Conclusion Metformin can inhibit the activation of PI3K/AKT signaling pathway by suppressing Gal-1 expression in intrahepatic cholangiocarcinoma, which in turn leads to the inhibition of cell proliferation.
关键词
二甲双胍 /
肝内胆管癌 /
Gal-1 /
PI3K/AKT
Key words
metformin /
intrahepatic cholangiocarcinoma /
Gal-1 /
PI3K/AKT
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参考文献
[1] Jia X, Lu S, Zeng Z, et al.Characterization of Gut Microbiota, Bile Acid Metabolism, and Cytokines in Intrahepatic Cholangiocarcinoma[J]. Hepatology, 2020, 71(3) : 893-906.
[2] Wang G, Wang Q, Liang N, et al.Oncogenic driver genes and tumor microenvironment determine the type of liver cancer[J]. Cell Death Dis, 2020, 11(5) : 313.
[3] Mallik R, Chowdhury TA.Metformin in cancer[J]. Diabetes Res Clin Pract, 2018, 143: 409-419.
[4] Marini C, Cossu V, Bauckneht M, et al.Metformin and Cancer Glucose Metabolism: At the Bench or at the Bedside?[J]. Biomolecules, 2021, 11(8) : 1231.
[5] Zhu M, Zhang P, Yu S, et al.Targeting ZFP64/GAL-1 axis promotes therapeutic effect of nab-paclitaxel and reverses immunosuppressive microenvironment in gastric cancer[J]. J Exp Clin Cancer Res, 2022, 41(1) : 14.
[6] Razumilava N, Gores GJ.Cholangiocarcinoma[J]. Lancet , 2014, 383(9935) : 2168-2179.
[7] 廖周俊, 杨少华, 刘立鑫, 等. AK4对肝内胆管癌细胞HUCCT1增殖, 迁移的影响[J]. 昆明医科大学学报, 2022, 43(6) : 6.
[8] El-Diwany R, Pawlik TM, Ejaz A.Intrahepatic Cholangiocarcinoma[J]. Surg Oncol Clin N Am, 2019, 28(4) : 587-599.
[9] Foretz M, Guigas B, Bertrand L, et al.Metformin: From Mechanisms of Action to Therapies[J]. Cell Metab, 2014, 20(6) : 953-966.
[10] Naa B, Fi C, Ag A, et al.Mechanisms of action of metformin in type 2 diabetes: Effects on mitochondria and leukocyte-endothelium interactions - ScienceDirect[J]. Redox Biol, 2020, 34: 101517.
[11] Vallianou N G, Evangelopoulos A, Kazazis C.Metformin and cancer.[J]. Rev Diabet Stud, 2013, 10(4) : 228-235.
[12] Li B, Zhou P, Xu K, et al.Metformin induces cell cycle arrest, apoptosis and autophagy through ROS/JNK signaling pathway in human osteosarcoma[J]. Int J Biol Sci, 2020, 16(1) : 74-84.
[13] Wu Y, Zheng Q, Li Y, et al.Metformin targets a YAP1-TEAD4 complex via AMPKα to regulate CCNE1/2 in bladder cancer cells[J]. J Exp Clin Cancer Res, 2019, 38(1) .
[14] Wang J L, Lan Y W, Tsai Y T, et al.Additive Antiproliferative and Antiangiogenic Effects of Metformin and Pemetrexed in a Non-Small-Cell Lung Cancer Xenograft Model[J]. Fron Cell Dev Biol, 2021, 9: 88062.
[15] Fryk E, Silva VRR, Jansson PA.Galectin-1 in Obesity and Type 2 Diabetes[J]. Metabolites, 2022, 12(10) : 930.
[16] Sideras K, Biermann K, Verheij J, et al.PD-L1, Galectin-9 and CD8+ tumor-infiltrating lymphocytes are associated with survival in hepatocellular carcinoma[J]. Oncoimmunology, 2017, 6(2) : e1273309.
[17] Kong F, Jin M, Cao D, et al.Galectin-3 not Galectin-9 as a candidate prognosis marker for hepatocellular carcinoma[J]. PeerJ, 2020, 8: e9949.
[18] Li H, Li J, Xiao W, et al.The Therapeutic Potential of Galectin-3 in the Treatment of Intrahepatic Cholangiocarcinoma Patients and Those Compromised With COVID-19[J]. Front Mol Biosci, 2021, 8: 666054.
[19] Cedeno-Laurent F, Dimitroff C J.Galectin-1 research in T cell immunity: Past, present and future[J]. Clin Immunol, 2012, 142(2) : 107-116.
[20] Song F, Hu B, Cheng JW, et al.Anlotinib suppresses tumor progression via blocking the VEGFR2/PI3K/AKT cascade in intrahepatic cholangiocarcinoma[J]. Cell Death Dis, 2020, 11(7) : 573.
[21] Zhang PF, Li KS, Shen YH, et al.Galectin-1 induces hepatocellular carcinoma EMT and sorafenib resistance by activating FAK/PI3K/AKT signaling[J]. Cell Death Dis, 2016, 7(4) : e2201.
基金
湖南省肝胆胰肠诊疗能力提升项目（湘卫医政医管处[2019]118号）
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