青蒿素抑制胆囊癌细胞的迁移侵袭及其机制

Effect and mechanism of artemisinin on inhibiting metastasis and invasion of gallbladder carcinoma cells

目的探讨青蒿素对胆囊癌细胞体外迁移、侵袭能力的影响及其机制。方法20μmol/L青蒿素处理胆囊癌细胞GBC-SD(青蒿素组),同时设立未加青蒿素处理的对照组。采用划痕实验检测胆囊癌细胞迁移能力变化,Transwell实验检测细胞侵袭能力变化,免疫荧光技术检测细胞形态变化。采用qRT-PCR和Western blot检测细胞中E-cadherin、Slug、Vimentin、MMP-2、MMP-9等基因的m RNA和蛋白水平变化。两组表达量比较采用t检验。结果划痕24 h青蒿素组划痕宽(393±23)μm,明显大于对照组的(210±15)μm(t=13.415,P<0.05)。青蒿素组GBC-SD穿膜细胞数为(93±8)个,明显少于对照组的(202±14)个(t=-17.037,P<0.05)。青蒿素促使细胞形态从间质型向表皮型转化。青蒿素组GBC-SD细胞的Slug、Vimentin、MMP-2、MMP-9基因的mRNA分别为0.89±0.03、1.35±0.10、3.30±0.13、1.15±0.08,明显低于对照组的2.08±0.12、3.22±0.15、4.72±0.19、1.95±0.15(t=-19.812,-20.775,-12.259,-9.438;P<0.05);而E-cadherin mRNA为2.06±0.08,明显高于对照组的1.03±0.06(t=20.956;P<0.05)。Western blot检测显示青蒿素下调Slug、Vimentin、MMP-2、MMP-9,上调E-cadherin蛋白。结论青蒿素可抑制胆囊癌细胞迁移和侵袭,其作用可能与抑制Slug基因表达逆转上皮间质转化,进而降低MMP表达有关。

Objective To investigate the effect of artemisinin on the in vitro metastasis and invasion of gallbladder carcinoma cells and its mechanism. Methods Gallbladder carcinoma cells GBCSD was treated with 20 μmol/L artemisinin（artemisinin group） and cells without artemisinin treatment was established as control group. The metastasis ability of gallbladder carcinoma cell was detected using scratch wound assay. The variation of cell invasion was evaluated using Transwell assay. The change of cell morphology was observed using immunofluorescent staining. The mRNA and protein level changes of E-cadherin, Slug, Vimentin, MMP-2 and MMP-9 were detected by qRT-PCR and Western blot. The expression levels between two groups were compared by t test.Results At 24 h,the scratch width was（393±23）μm in artemisinin group,significantly wider than（210±15）μm in control group（t=13.415,P〈0.05）.In artemisinin group,the quantity of transmembrane GBC-SD cells was 93±8,significantly less than 202±14in control group（t=-17.037,P〈0.05）.Artemisinin promoted the epithelial-mesenchymal transition of cell morphology.In artemisinin group,the expression levels of Slug,Vimentin,MMP-2 and MMP-9 mRNA of GBC-SD cells were 0.89±0.03,1.35±0.10,3.30±0.13 and 1.15±0.08,significantly lower than 2.08±0.12,3.22±0.15,4.72±0.19 and 1.95±0.15 in control group（t=-19.812,-20.775,-12.259,-9.438;P〈0.05）.In artemisinin group,the expression of E-cadherin mRNA was 2.06±0.08,significantly higher than 1.03±0.06 in control group（t=20.956;P〈0.05）.Western blot showed that artemisinin could down-regulate the expression levels of Slug,Vimentin,MMP-2 and MMP-9 proteins,whereas up-regulate the expression of E-cadherin protein.Conclusions Artemisinin can inhibit the metastasis and invasion of gallbladder carcinoma cells.It is probably related with suppressing the expression of Slug gene,reversing the epithelial-mesenchymal transition,thereby down-regulating the expression level of MMP.