烟酰胺对鼻咽癌CNE-1细胞增殖、凋亡与迁移作用机制研究

Effects and mechanism of nicotinamide on proliferation,apoptosis and migration of nasopharyngeal carcinoma CNE-1 cells

目的探讨烟酰胺对人鼻咽癌CNE-1细胞增殖、凋亡及迁移的影响和机制。方法将人鼻咽癌CNE-1细胞株分为烟酰胺处理组(10、20、40、80μmol/L烟酰胺)及对照组。CCK-8法检测不同浓度烟酰胺作用48、72 h后CNE-1细胞增殖率,Annexin V-FITC/PI双染法流式细胞术检测40μmol/L烟酰胺作用48 h后CNE-1细胞凋亡率和细胞周期,Transwell小室实验检测CNE-1细胞迁移及侵袭能力的变化,实时荧光定量PCR和Western blot分别检测CNE-1细胞中p53蛋白和基质金属蛋白酶9(MMP-9)mRNA和蛋白水平。结果与对照组相比,CCK-8法检测结果显示,烟酰胺处理组CNE-1细胞增殖抑制率升高(P<0.01),且随着烟酰胺处理浓度的升高和处理时间的延长而升高,呈浓度和时间依赖性;40μmol/L烟酰胺处理CNE-1细胞48 h(即40μmol/L实验组)后,CNE-1细胞凋亡率升高(P<0.01);40μmol/L实验组G2/M期细胞比例增加,G_(0)/G_(1)期细胞比例降低(P<0.05);CNE-1细胞经40μmol/L烟酰胺处理后其侵袭及迁移能力均下降(P<0.01);40μmol/L实验组p53蛋白表达水平升高,MMP-9蛋白表达水平下降。结论烟酰胺可通过上调p53蛋白的表达、恢复细胞周期从而抑制人鼻咽癌CNE-1细胞增殖,促进其凋亡,同时亦可影响MMP-9蛋白的表达进而降低CNE-1细胞迁移和侵袭能力。

Objective To investigate the effect and potential mechanism of nicotinamide on proliferation,apoptosis and migration of nasopharyngeal carcinoma(NPC)CNE-1 cells.Methods NPC CNE-1 cells were divided into blank control group and the experimental groups,which were treated with 10,20,40 and 80μmol/L of nicotinamide respectively,while the blank control was treated without nicotinamide.The proliferation rate of CNE-1 cells was detected by CCK-8 after 48 h and 72 h treatment with the concentrations mentioned above,the apoptosis rates and the cell cycle retardation of CNE-1 cells treated with 40μmol/L nicotinamide for 48 h were detected by Annexin V-FITC/PI double staining flow cytometry.Cell migration and invasion were evaluated by transwell assay.Real-time fluorescent quantitative PCR and Western blot were used to detect the mRNA level and the expression of p53 and matrix metalloproteinases 9(MMP-9)proteins in CNE-1 cells.Results Compared with the blank control group,the data of CCK-8 assay showed that nicotinamide had an effective inhibition on the proliferation activity of CNE-1 cells(P<0.01),and the effect was in time-dose dependence manner.Flow cytometry results showed that the nicotinamide could promote apoptosis of CNE-1 cells treated with 40μmol/L nicotinamide for 48 h(P<0.01),with the number of cells increased in G2/M and decreased in G_(0)/G_(1) phase(P<0.05).And the migration and invasion were inhibited.Compared with the control group,the level of p53 protein was upregulated,while the level of MMP-9 protein decreasedwith 40μmol/L nicotinamide treatment for 48 h.Conclusion Nicotinamide can inhibit the proliferation and promote the apoptosis of human nasopharyngeal carcinoma CNE-1 cells by up-regulating the expression of p53 protein and restoring the cell cycle,and also affect the expression of MMP-9 protein,thus reducing the migration and invasion ability of CNE-1 cells.