核因子-κB/p65 siRNA介导的舌鳞状细胞癌Tca8113细胞凋亡及相关分子机制

NF-κB p65 siRNA Mediated Cell Apoptosis of Tongue Squamous Cell Carcinoma Tca8113 Cells and its Related Molecular Mechanism

目的:研究下调核因子κB(nuclear factor-kappa B,NF-κB)/p65基因的表达对舌鳞状细胞癌Tca8113细胞凋亡的影响,并探讨Tca8113细胞凋亡可能的分子机制。方法:利用脂质体2000将终浓度为100nmol/L的p65 siRNA转染舌鳞状细胞癌Tca8113细胞,通过RT-PCR检测0、24、48和72hp65 mRNA的表达,并通过流式细胞术检测转染前后细胞凋亡变化,通过Caspase-Glo○R-3/7,-8和-9检测试剂盒分析caspase-3/9活性,用Western blotting技术检测p65、bcl-2和bax蛋白表达。结果:Tca8113细胞转染p65siRNA后,在48hp65 mRNA的相对表达量为0.181±0.028,显著低于未处理组0.595±0.038和对照siRNA组0.613±0.067(P<0.05)。流式细胞术结果显示,p65 siRNA能促使Tca8113细胞凋亡,其早期凋亡比率为(23.16±1.72)%,显著高于未处理组和对照siRNA组(P<0.01)。转染48h后,p65和bcl-2蛋白的相对表达水平明显下调,而促凋亡蛋白bax的表达显著上升,并伴随着caspase-3/9活性的显著上升。结论:NF-κB信号途径中的p65亚基可能在舌鳞状细胞癌凋亡中发挥重要作用,该研究有望为舌鳞状细胞癌的分子靶向治疗提供理论依据。

Objective： To investigate effect of downregulation of p65 gene on cell apoptosis and explore the possible molecular mechanism of cell apoptosis in tongue squamous cell carcinoma cell line Tca8113 cells.Methods： Tca8113 cells were transfected with 100 nM p65 siRNA.The expression level of p65 mRNA was measured using RT-PCR at 0h,24h,48h and 72h.Meanwhile,cell apoptosis was detected using flow cytometry.Activities of caspase-3/9 was detected by Caspase-Glo○R-3/7,-8 and-9 kit,and expressions of p65,bcl-2 and bax proteins were determined using Western blotting.Results： The relative expression level of p65 mRNA in Tca8113 cells reached the lowest 48 h after transfected with p65 siRNA,and the relative expression level（0.181±0.028） in p65 siRNA group was significantly lower than that in untreated group（0.595±0.038） or control siRNA group（0.613±0.067）（P0.05）.The results of flow cytometry demonstrated that p65 siRNA could induce Tca8113 cells apoptosis,and early cell apoptosis ratio（23.16±1.72%） in p65 siRNA group was significantly higher than that in untreated group or control siRNA group（P0.01）.The protein levels of p65 and bcl-2 obviously decreased,and the bax protein level and activities of caspase-3/9 significantly increased 48 h after transfected with p65 siRNA.Conclusion： p65 subunit in NF-κB signaling pathway play a pivotal role in cell apoptosis of tongue squamous cell carcinoma,which will provide theoretical basis for molecular target therapy of tongue squamous cell carcinoma.