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石蒜碱对胃癌AGS细胞恶性生物学行为的影响及机制研究▲
Effects of lycorine on malignant biological behaviors of AGS gastric cancer cells and its mechanism

内科 202419卷04期 页码:363-371

作者机构:1 广西医科大学第一附属医院消化内科,南宁市 530021;2 广西医科大学第二附属医院消化内科,南宁市 530007

基金信息:广西医疗卫生适宜技术开发与推广应用(S2019100) 通信作者:赖铭裕

DOI:10.16121/j.cnki.cn45⁃1347/r.2024.04.03

  • 中文简介
  • 英文简介
  • 参考文献

目的 探讨石蒜碱对胃癌AGS细胞恶性生物学行为的影响并初步探讨其可能的作用机制。方法 用不同浓度的盐酸石蒜碱(LH)(0 μmol/L、0.015 μmol/L、0.062 5 μmol/L、0.25 μmol/L、1 μmol/L、4 μmol/L、16 μmol/L)干预AGS细胞0 h、24 h和48 h,采用细胞计数试剂盒-8(CCK-8)试验计算干预48 h时药物半数抑制浓度(IC50),以确定后续试验的LH浓度。将AGS细胞分为对照组(0.1%二甲基亚砜处理)、LH-L组(0.125 μmol/L LH处理)、LH-M组(0.5 μmol/L LH处理),LH-H组(2 μmol/L LH处理),以及微小RNA(miRNA)-675-nc组(转染慢病毒空白载体)、miRNA-675-mimic组(转染慢病毒miRNA-675过表达载体)和LH+miRNA-675-mimic组(转染慢病毒miRNA-675过表达载体+2 μmol/L LH处理)。应用CCK-8试验、平板克隆试验、细胞划痕愈合试验和Transwell试验分别检测各组细胞的增殖、迁移和侵袭能力;应用实时荧光定量PCR检测miRNA-675和糖原合成酶激酶(GSK)-3β mRNA的相对表达水平;应用蛋白质印迹法检测GSK-3β和β-联蛋白(β-catenin)蛋白相对表达水平。结果 在0.015~16 μmol/L范围内,LH可时间-浓度依赖地抑制AGS细胞的增殖能力(P<0.05),干预48 h时,药物IC50为(0.81±0.13)μmol/L。对照组、LH-L组、LH-M组、LH-H组干预14 d后细胞克隆数,干预24 h、48 h后划痕愈合率,干预24 h后侵袭细胞数、迁移细胞数,干预48 h后miRNA-675相对表达水平均依次降低;干预48 h后LH-M组细胞的GSK-3β mRNA相对表达水平高于对照组,LH-H组细胞的GSK-3β mRNA相对表达水平高于对照组、LH-L组、LH-M组;干预48 h后LH-M组GSK-3β蛋白相对表达水平高于对照组,LH-H组GSK-3β蛋白相对表达水平高于对照组和LH-L组;干预48 h后LH-H组β-catenin蛋白相对表达水平低于对照组和LH-L组(均P<0.05)。miRNA-675-mimic组的光密度值(48 h)、细胞克隆数(14 d)、细胞侵袭数(24 h)、细胞迁移数(24 h)、β-catenin蛋白相对表达水平(48 h)均高于miRNA-675-nc组、LH+miRNA-675-mimic组;miRNA-675-mimic组GSK-3β蛋白相对表达水平(48 h)低于miRNA-675-nc组、LH+miRNA-675-mimic组(均P<0.05)。结论 石蒜碱能有效地抑制胃癌AGS细胞增殖、侵袭和迁移能力,该作用可能通过调控miRNA-675/GSK-3β/β-catenin轴实现。

Objective To investigate the effects of lycorine on the malignant biological behaviors of AGS gastric cancer cells, and to preliminarily explore its possible action mechanism. Methods AGS cells were intervened with different concentrations of lycorine hydrochloride (LH) (0 μmol/L, 0.015 μmol/L, 0.062,5 μmol/L, 0.25 μmol/L, 1 μmol/L, 4 μmol/L, 16 μmol/L) for 0 hour, 24 hours, and 48 hours, and its inhibitory concentration 50 (IC50) at the 48th hour of the intervention was calculated by the cell counting kit-8 (CCK-8) test to determine the LH concentration in subsequent trials. AGS cells were divided into a control group (treated with 0.1% dimethyl sulfoxide), an LH-L group (treated with 0.125 μmol/L LH), an LH-M group (treated with 0.5 μmol/L LH), or an LH-H group (treated with 2 μmol/L LH), as well as a microRNA (miRNA)-675-nc group (transfected with blank lentiviral vectors), a miRNA-675-mimic group (transfected with lentiviral vectors overexpressing miRNA-675), or an LH+miRNA-675-mimic group (transfected with lentiviral vectors overexpressing miRNA-675 + treated with 2 μmol/L LH). CCK-8 test, clonogenic assay in plates, cell scratch healing assay, and Transwell test were separately used to detect the proliferation, migration, and invasion abilities of cells in each group; real-time fluorogenic quantitative PCR was used to detect the relative expression levels of miRNA-675 and glycogen synthase kinase (GSK)-3β mRNA; western blotting was used to detect the relative protein expression levels of GSK-3β and β-catenin. Results In the range of 0.015-16 μmol/L, LH could inhibit the proliferation ability of AGS cells in a time-concentration dependent manner (P<0.05), and its IC50 was (0.81±0.13) μmol/L at the 48th hour of the intervention. The number of cell colonies after 14 days of intervention, the scratch healing rates 24 hours and 48 hours after the intervention, the numbers of invasive cells and migrated cells 48 hours after the intervention, and the relative expression level of miRNA-675 48 hours after the intervention in the control group, LH-L group, LH-M group, and LH-H group decreased successively; 48 hours after the intervention, the relative expression level of GSK-3β mRNA was higher in the LH-M group than in the control group, and the relative expression level of GSK-3β mRNA was higher in the LH-H group than in the control group, LH-L group, and LH-M group; 48 hours after the intervention, the relative protein expression level of GSK-3β was higher in the LH-M group than in the control group, and the relative protein expression level of GSK-3β was higher in the LH-H group than in the control group and LH-L group; 48 hours after the intervention, the relative protein expression level of β-catenin was lower in the LH-H group than in the control group and LH-L group (all P<0.05). The optical density (48 hours), number of cell colonies (14 days), numbers of invasive cells and migrated cells (24 hours), and relative protein expression level of β-catenin (48 hours) were higher in the miRNA-675-mimic group than in the miRNA-675-nc group and LH+miRNA-675-mimic group; the relative protein expression level of GSK-3β (48 hours) was lower in the miRNA-675-mimic group than in the miRNA-675-nc group and LH+miRNA-675-mimic group (all P<0.05). Conclusion Lycorine can effectively inhibit the proliferation, invasion, and migration abilities of AGS gastric cancer cells, which may be achieved by its regulation on the miRNA-675/GSK-3β/β-catenin axis.


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