Influence of pulsatilla saponin on proliferation, migrationand invasion of gastric cancer HGC-27 cells and its possible mechanism
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摘要:目的
探讨白头翁皂苷对胃癌细胞HGC-27增殖、迁移和侵袭的影响及其可能机制。
方法采用不同浓度白头翁皂苷(12.5、25.0、50.0 μmol/L)处理人胃癌细胞HGC-27, 分别设为低白头翁皂苷组、中白头翁皂苷组、高白头翁皂苷组，另将正常培养的HGC-27细胞设为对照组。将si-NC、si-circNRIP1、pcDNA、pcDNA-circNRIP1转染至HGC-27细胞，分别设为si-NC组、si-circNRIP1组、pcDNA组、pcDNA-circNRIP1组。向HGC-27细胞中转染pcDNA、pcDNA-circNRIP1, 均加入50.0 μmol/L白头翁皂苷培养，分别记为白头翁皂苷+pcDNA组、白头翁皂苷+pcDNA-circNRIP1组。通过CCK-8法、平板克隆形成实验和Transwell实验分别检测细胞增殖、克隆形成和迁移、侵袭能力; 采用实时荧光定量聚合酶链反应(qRT-PCR)法检测环状RNA核受体相互作用蛋白1 (circNRIP1)表达量; 采用蛋白质印迹法(Western blot)检测E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)的蛋白表达情况。
结果对照组、低白头翁皂苷组、中白头翁皂苷组、高白头翁皂苷组细胞增殖抑制率、E-cadherin蛋白水平依次升高，细胞克隆形成数量、迁移数量、侵袭数量依次减少, N-cadherin蛋白水平、circNRIP1表达量依次降低，差异均有统计学意义(P < 0.05); pcDNA-circNRIP1组circNRIP1表达量高于pcDNA组, si-circNRIP1组circNRIP1表达量低于si-NC组，差异有统计学意义(P < 0.05); si-circNRIP1组细胞增殖抑制率和E-cadherin蛋白水平高于si-NC组, N-cadherin蛋白水平低于si-NC组，细胞克隆形成数量、迁移数量和侵袭数量少于si-NC组，差异均有统计学意义(P < 0.05); 白头翁皂苷+pcDNA-circNRIP1组细胞增殖抑制率和E-cadherin蛋白水平低于白头翁皂苷+pcDNA组, N-cadherin蛋白水平高于白头翁皂苷+pcDNA组，细胞克隆形成数量、迁移数量和侵袭数量多于白头翁皂苷+pcDNA组，差异有统计学意义(P < 0.05)。
结论白头翁皂苷可抑制胃癌细胞增殖、迁移、侵袭活性且呈现浓度依赖性，其作用机制或与下调circNRIP1表达有关。
Abstract:ObjectiveTo explore the influence of pulsatilla saponin on proliferation, migration and invasion of gastric cancer HGC-27 cells and its possible mechanism.
MethodsHuman gastric cancer HGC-27 cells were treated with different doses of pulsatilla saponin (12.5, 25.0, 50.0 μmol/L) and named as low-dose pulsatilla saponin group, medium-dose pulsatilla saponin group and high-dose pulsatilla saponin group, and the normal cultured HGC-27 cells were named as control group. The si-NC, si-circNRIP1, pcDNA and pcDNA-circNRIP1 were transfected into HGC-27 cells, and were named as si-NC group, si-circNRIP1 group, pcDNA group and pcDNA-circNRIP1 group respectively. HGC-27 cells were transfected with pcDNA and pcDNA-circNRIP1 and cultured with 50.0 μmol/L pulsatilla saponin, and were named as pulsatilla saponin plus pcDNA group and pulsatilla saponin plus pcDNA-circNRIP1 group respectively. CCK-8 assay, colony formation assay and Transwell assay were used to detect the proliferation, clone formation and migration, and invasion respectively; quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of circular RNA nuclear receptor interacting protein 1 (circNRIP1); Western blot was used to detect the expressions of E-cadherin and N-cadherin proteins.
ResultsIn the control group, low-dose pulsatilla saponin group, medium-dose pulsatilla saponin group and high-dose pulsatilla saponin group, the inhibition rates of cell proliferation and the levels of E-cadherin protein significantly increased gradually, the number of cell clone formation, migration and invasion significantly decreased gradually, and the expressions of N-cadherin protein and circNRIP1 significantly decreased gradually (P < 0.05); the expression level of circNRIP1 in the pcDNA-circNRIP1 group was significantly higher than that in the pcDNA group, while the expression level of circNRIP1 in the si-circNRIP1 group was significantly lower than thatin the si-NC group (P < 0.05); the inhibition rate of cell proliferation and level of E-cadherin protein in the si-circNRIP1 group were significantly higher than those in the si-NC group, while the level of N-cadherin protein and the number of cell clone formation, migration and invasion were significantly lower than those in the si-NC group (P < 0.05); the inhibition rate of cell proliferation and level of E-cadherin protein in the pulsatilla saponin plus pcDNA-circNRIP1 group were significantly lower than those in the pulsatilla saponin plus pcDNA group, while the level of N-cadherin protein and the number of cell clone formation, migration and invasion were significantly higher than those in the pulsatilla saponin plus pcDNA group (P < 0.05).
ConclusionPulsatilla saponin can inhibit the proliferation, migration and invasion of gastric cancer cells in a dose-dependent manner, and its mechanism may be associated with down-regulation of circNRIP1 expression.
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图 2 4组细胞Transwell实验结果和E-cadherin、N-cadherin蛋白表达情况
A: Transwell迁移和侵袭实验结果(结晶紫染色法，放大200倍); B: E-cadherin、N-cadherin蛋白的Western blot检测结果。
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图 3 抑制circNRIP1对HGC-27细胞增殖、迁移、侵袭能力的影响
A: 平板克隆形成实验结果; B: Transwell迁移和侵袭实验结果(结晶紫染色法，放大200倍); C: E-cadherin、N-cadherin的Western blot检测结果。
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图 4 过表达circNRIP1对白头翁皂苷处理的HGC-27细胞增殖、迁移、侵袭的影响
A: 平板克隆形成实验结果; B: Transwell迁移和侵袭实验结果(结晶紫染色法，放大200倍); C: E-cadherin、N-cadherin的Western blot检测结果。
表 1 4组细胞增殖抑制率和细胞克隆形成数量比较(x±s)
组别 n 细胞增殖抑制率/% 细胞克隆形成数量/个 对照组 9 0±0 103.89±6.92 低白头翁皂苷组 9 10.50±1.06* 85.56±3.50* 中白头翁皂苷组 9 23.51±1.29*# 63.11±2.85*# 高白头翁皂苷组 9 44.71±2.37*#△ 42.44±2.31*#△ 与对照组比较, *P < 0.05; 与低白头翁皂苷组比较, #P < 0.05; 与中白头翁皂苷组比较, △P < 0.05。 
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表 2 4组细胞迁移、侵袭数量和E-cadherin、N-cadherin蛋白表达情况比较(x±s)
组别 n 迁移数量/个 侵袭数量/个 E-钙黏蛋白 N-钙黏蛋白 对照组 9 221.56±11.92 163.00±7.27 0.22±0.03 0.89±0.06 低白头翁皂苷组 9 182.00±7.42* 132.67±5.12* 0.38±0.04* 0.64±0.05* 中白头翁皂苷组 9 149.78±6.41*# 98.00±4.35*# 0.56±0.06*# 0.43±0.04*# 高白头翁皂苷组 9 105.11±5.13*#△ 70.33±3.53*#△ 0.77±0.06*#△ 0.21±0.02*#△ 与对照组比较, *P < 0.05; 与低白头翁皂苷组比较, #P < 0.05; 与中白头翁皂苷组比较, △P < 0.05。
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表 3 4组细胞circNRIP1表达量比较(x±s)
组别 n circNRIP1 对照组 9 1.00±0 低白头翁皂苷组 9 0.71±0.05* 中白头翁皂苷组 9 0.48±0.05*# 高白头翁皂苷组 9 0.22±0.02*#△ circNRIP1: 环状RNA核受体相互作用蛋白1。与对照组比较, *P < 0.05; 与低白头翁皂苷组比较, #P < 0.05; 与中白头翁皂苷组比较, △P < 0.05。
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表 4 各组circNRIP1转染效率检测结果比较(x±s)
组别 n circNRIP1 pcDNA组 9 1.00±0 pcDNA-circNRIP1组 9 2.71±0.10* si-NC组 9 1.00±0 si-circNRIP1组 9 0.46±0.05# 与pcDNA组比较, *P < 0.05; 与si-NC组比较, #P < 0.05。
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表 5 抑制circNRIP1对HGC-27细胞增殖、迁移、侵袭相关指标的影响(x±s)
组别 n 细胞增殖抑制率/% 细胞克隆形成数量/个 迁移数量/个 侵袭数量/个 E-cadherin蛋白水平 N-cadherin蛋白水平 si-NC组 9 0±0 102.56±9.27 218.00±10.31 165.00±11.22 0.21±0.03 0.85±0.07 si-circNRIP1组 9 36.38±2.15* 53.11±2.88* 125.00±8.03* 85.78±5.53* 0.64±0.06* 0.32±0.03* 与si-NC组比较, *P < 0.05。
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表 6 过表达circNRIP1对白头翁皂苷处理的HGC-27细胞增殖、迁移、侵袭相关指标的影响(x±s)
组别 n 细胞增殖抑制率/% 细胞克隆形成数量/个 迁移数量/个 侵袭数量/个 E-cadherin蛋白水平 N-cadherin蛋白水平 白头翁皂苷+pcDNA组 9 44.36±1.94 41.89±3.14 106.44±7.24 72.00±4.97 0.76±0.07 0.19±0.03 白头翁皂苷+pcDNA-circNRIP1组 9 15.53±1.20* 79.44±2.71* 162.11±9.47* 114.56±6.24* 0.41±0.05* 0.53±0.05* 与白头翁皂苷+pcDNA组比较, *P < 0.05。
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