摘要目的 探讨辛伐他汀对缺氧黑素瘤细胞A375增殖、凋亡、迁移调控的分子机制.方法 在常氧或缺氧环境下体外培养A375细胞,细胞分为辛伐他汀处理的辛伐他汀组与二甲基亚砜处理的对照组,通过CCK-8细胞计数试剂盒检测细胞活力,Annexin V/PI凋亡实验检测细胞凋亡,插入式细胞培养皿transwell迁移实验检测细胞迁移.RT-PCR法、Western印迹法检测辛伐他汀组与对照组细胞低氧诱导因子(HIF)-1α、生存素、细胞周期素依赖性蛋白激酶抑制因子(P27),以及沉默缺氧A375细胞的HIF-1α后的生存素、p27 mRNA与蛋白表达水平.结果 常氧或缺氧环境下,辛伐他汀组与对照组A375细胞增殖活力、凋亡细胞比例、细胞迁移数组间差异均有统计学意义(F值95.84、37.22、177.5,均P＜0.001),缺氧对照组A375细胞的增殖活力(1.391±0.129)、细胞迁移数(322.550±26.226)均高于常氧对照组(0.807±0.049、125.583±17.256)、缺氧辛伐他汀组(0.685±0.417、115.167±12.050)(P＜ 0.001);缺氧对照组细胞凋亡比例(6.167％±2.714％)均低于常氧对照组(11.833％±2.483％)、缺氧辛伐他汀组(17.833％±2.714％)(P＜0.01).缺氧辛伐他汀组HIF-1αmRNA与蛋白水平、生存素mRNA与蛋白水平均低于对照组(P＜ 0.05);P27mRNA与蛋白水平均高于对照组(P＜0.05).沉默缺氧A375细胞中HIF-1α后,生存素mRNA与蛋白水平均低于对照组(t值为5.346、8.281,P＜0.05),P27 mRNA与蛋白水平高于对照组(t值为31.37、9.954,P＜0.01).结论 辛伐他汀可抑制缺氧A375细胞的增殖及迁移,促进凋亡,其机制可能与HIF通路有关.

abstractsObjective To evaluate the regulatory effect of simvastatin on the proliferation,apoptosis and migration of hypoxic A375 melanoma cells,and to explore their molecular mechanisms.Methods Under normoxic or hypoxic culture conditions,A375 cells were treated with 0.25,0.5,1,2,4,8 μmol/L simvastatin (simvastatin groups) and dimethyl sulfoxide (DMSO,control group) separately for 48 hours.Cell counting kit-8 (CCK-8) assay,Transwell assay using immersion culture system and flow cytometry using annexin-V/propidium iodide (PI) staining were conducted to assess the proliferative and migratory activities and apoptosis of A375 cells,respectively.RT-PCR and Western blot analysis were performed to measure the mRNA and protein expression of hypoxia-inducible factor 1oα (HIF-1α),survivin and cyclin-dependent kinase inhibitor p27 in the simvastatin groups and control group,as well as in the HIF-1α-silenced hypoxic A375 cells.Results Under normoxic or hypoxic culture conditions,there were significant differences in the proliferative activities of A375 cells,proportion of apoptotic cells and number of migrating cells between the simvastatin groups and control groups (F =95.84,37.22 and 177.5 respectively,P ＜ 0.001).The hypoxic control group showed significantly higher cellular proliferative activity (1.391 ± 0.129) and higher number of migrating cells (322.550 ± 26.226) compared with the normoxic control group (0.807 ± 0.049,125.583 ± 17.256 respectively,both P ＜ 0.001) and hypoxia + simvastatin group (0.685 ± 0.417,115.167 ± 12.050 respectively,both P ＜ 0.001),but significantly lower proportion of apoptotic cells (6.167％ ± 2.714％) compared with the normoxic control group (11.833％ ± 2.483％,P ＜ 0.01) and hypoxia + simvastatin group (17.833％ ± 2.714％,P ＜ 0.01).Under the hypoxic condition,the simvastatin group showed significantly lower mRNA and protein expression of HIF-1α and survivin compared with the control group (all P ＜ 0.05),but significantly higher mRNA and protein expression of P27 compared with the control group (both P ＜ 0.05).Under the hypoxic condition,HIF-1α-silenced A375 cells showed significantly decreased mRNA and protein expression of survivin (t =5.346 and 8.281 respectively,both P ＜ 0.05),but significantly increased mRNA and protein expression of p27 (t =31.37 and 9.954 respectively,both P ＜ 0.01) compared with the unsilenced cells.Conclusion Simvastatin can inhibit the proliferation and migration of hypoxic A375 cells,and promote their apoptosis,likely by activating the HIF signaling pathway.