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目的:观察莱菔硫烷(Sulforaphane,SFN)对宫颈癌细胞迁移和侵袭的抑制情况,并探讨其分子机制。方法:体外培养宫颈癌细胞系HeLa细胞,经100 nmol/L佛波脂PMA诱导24 h后,加入不同浓度的SFN继续孵育24 h。MTT法检测HeLa细胞的生长抑制情况;DCF染色法检测细胞内活性氧(Reactive oxygen species,ROS)的产生,细胞伤口愈合试验和小室侵袭实验分析SFN对PMA诱导HeLa细胞的迁移与侵袭情况。提取细胞总RNA,逆转录PCR检测基质金属蛋白酶-9(matrix met-alloproteinase-9,MMP-9)的表达,明胶酶谱实验检检测其酶活性。萤光素酶报告基因法检测SFN对核因子κB(Nuclear Fac-tor kappa B,NF-κB)活性的影响。结果:0～30μmol/L SFN对HeLa细胞的生长增殖无明显毒性作用。DCF染色显示PMA处理能使HeLa细胞ROS的含量增高5倍,而30μmol/L SFN处理能使ROS的产生降低44%;SFN也能使HeLa细胞迁移和侵袭率分别降低60%和75.5%。RT-PCR结果显示SFN能以剂量依赖性方式抑制PMA诱导的MMP-9表达,明胶酶谱实验显示MMP-9酶活性也明显降低。报告基因实验显示SFN能以剂量依赖性方式抑制NF-κB的活性。结论:SFN抑制NF-κB介导的MMP-9表达,从而影响宫颈癌细胞的迁移和侵袭。
Objective: To observe the inhibitory effect of sulforaphane (SFN) on the migration and invasion of cervical cancer cells and to explore its molecular mechanism. Methods: HeLa cells were cultured in vitro and induced by PMA at 100 nmol / L for 24 h. SFN was added in different concentrations for 24 h. MTT assay was used to detect the growth inhibition of HeLa cells. DCF staining was used to detect the production of intracellular reactive oxygen species (ROS). Cell wound healing assay and cell invasion assay were used to analyze the migration and invasion of HeLa cells induced by PMA. Total RNA was extracted and the expression of matrix metalloproteinase-9 (MMP-9) was detected by reverse transcription polymerase chain reaction (RT-PCR). The enzyme activity was detected by gelatin zymography. The luciferase reporter assay was used to detect the effect of SFN on nuclear factor-κB (NF-κB) activity. Results: 0 ~ 30μmol / L SFN had no obvious toxic effect on the proliferation of HeLa cells. DCF staining showed that the PMA treatment could increase the content of ROS in HeLa cells by 5-fold, while the treatment with 30μmol / L SFN reduced the production of ROS by 44%. SFN also reduced the migration and invasion of HeLa cells by 60% and 75.5%, respectively. RT-PCR results showed that SFN could inhibit the PMA-induced MMP-9 expression in a dose-dependent manner. Gelatin zymography showed that MMP-9 activity was also significantly decreased. Reporter gene experiments show that SFN can inhibit NF-κB activity in a dose-dependent manner. Conclusion: SFN inhibits NF-κB-mediated MMP-9 expression and thus affects the migration and invasion of cervical cancer cells.