桔梗皂苷D对HCCLM3细胞迁移侵袭影响机制探讨

Migration and invasion effect of platycodin D for HCCLM3 cells

目的桔梗皂苷D(platycodin D,PD)是中药材桔梗的主要活性成分,对多种肿瘤细胞具有抗肿瘤活性。有研究发现,它能够促进肝癌细胞的凋亡和抑制增殖,但对细胞侵袭性的影响在很大程度上是未知的。本实验研究PD对人肝癌HCCLM3细胞迁移侵袭作用的影响,并初步探讨其可能的作用机制。方法不同浓度的PD处理肝癌HCCLM3细胞24和48h,MTT法检测对细胞增殖的影响;划痕实验和Transwell迁移侵袭实验检测对细胞迁移和侵袭的作用;荧光定量PCR和蛋白质印迹实验分别检测基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)和MMP-9基因在mRNA和蛋白水平的表达情况。结果 MTT检测结果表明,PD降低肝癌HCCLM3细胞的存活率,抑制细胞的增殖,并呈剂量依赖性,P<0.01。划痕实验结果表明,10、20和40μmol/L的PD处理细胞48h后,细胞的迁移率分别为(41.33±8.25)%、(31.87±9.83)%和(12.29±3.10)%,与对照组(86.53±6.04)%相比,差异有统计学意义,F=56.28,P<0.001。Transwell实验结果表明,10、20和40μmol/L的PD处理细胞48h后,迁移的细胞个数分别为466.33±103.94、294.67±42.53和156.68±52.27,与对照组818.33±108.15相比,差异有统计学意义,F=36.47,P<0.001;侵袭的细胞个数分别为299.33±92.93、192.67±55.63和64.00±28.90,与对照组665.67±76.84相比,差异有统计学意义,F=43.48,P<0.001。荧光定量PCR和蛋白质印迹实验进一步研究表明,10、20和40μmol/L的PD处理细胞后,MMP-2和MMP-9在mRNA水平的表达降低,与对照组相比,差异有统计学意义,F值分别为16.86和27.32,P值分别为0.002和0.001;同时,MMP-2和MMP-9在蛋白水平的表达也降低,F值分别为8.85和15.01,P值分别为0.006和0.001。结论 PD能够抑制人肝癌HCCLM3细胞的迁移和侵袭能力,可能与降低MMP-2和MMP-9的表达有关。

OBJECTIVE Platycodin D,the main saponin isolated from Chinese herb Platycodon grandiflorum,exhibits anticancer activities against various tumour cell lines.Previous studies have found it promoted apoptosis and inhibited proliferation of hepatocellular carcinoma（HCC）cells,but the effect on cell invasiveness is largely unknown.This study is to investigate the effect of platycodin D on migration and invasion on human HCC cell HCCLM3 and to primarily explore the possible mechanisms.METHODS HCC cell HCCLM3 treated with different concentrations of platycodin D for 24 h and 48 h,and then the effect of proliferation was detected by MTT.The effect of migration and invasion was detected by wound healing assay and transwell assay.The expression of MMP-2 and MMP-9 in mRNA and protein level was detected by qRT-PCR and western blot assay.RESULTS MTT assay showed that platycodin D reduced the survival rate and inhibited the proliferation of HCC cell HCCLM3 in a dose-dependent manner（P〈0.01）.Wound healing assay showed that the migration rates of cell treated with 10,20 and 40μmol/L platycodin D for 48 hwere（41.33±8.25）%,（31.87±9.83）% and（12.29±3.10）% vs in control group（86.53±6.04）%,F=56.28,P〈0.001.Transwell assay showed that the migration treated with 10,20 and 40μmol/L platycodin D for 48 h were 466.33±103.94,294.67±42.53 and156.68±52.27 vs in control group 818.33±108.15,F=36.47,P〈0.001,and invasion number of cells were 299.33±92.93,192.67±55.63 and 64.00±28.90 vs in control group 665.67±76.84,（F=43.48,P〈0.001）.Further studies showed that the expression of MMP-2 and MMP-7 RNA in cells detected by qRT-PCR was down-regulated,when the cells was treated with 10,20 and 40μmol/L of platycodin D,compared with the control group（F=16.86,P=0.002 and F=27.32,P〈0.001）at the same time,the expression of MMP-2 and MMP-7 protein in cells detected by western blot was down-regulated,compared with the control group（F=8.85,P=0.006 and F=15.01,P=0.001）.CONCLUSION Our findings show Platycodin D can significantly inhibit migration and invasion of human HCC HCCLM3 cells,which may be related to the down-regulated expression of MMP-2 and MMP-9.