NF-κB信号通路的阻断对皮肤鳞癌SCL-1细胞凋亡的影响

Effect of blockade of NF-κB signaling pathway on cell apoptosis in cutaneous squamous cell carcinoma

目的:利用siRNA技术抑制核因子-kappa B(nuclear factor-kappa B,NF-κB)亚单位p65基因的表达,研究其对p65表达的抑制作用,并探讨其对皮肤鳞癌SCL-1细胞凋亡的影响。方法:将终浓度为50 nmol/L的p65siR-NA转染皮肤鳞癌SCL-1细胞,通过RT-PCR检测p65mRNA的表达;利用Western blotting检测p65、bcl-2和bax蛋白表达,利用Caspase-Glo-3/7,8和9检测试剂盒检测caspase-3/9的活性;最后通过流式细胞术检测细胞凋亡。结果:p65siRNA转染SCL-1细胞后的48 h,p65mRNA的表达水平最低,与0 h相比,差异有统计学意义(0.23±0.10vs.0.66±0.05,P<0.05);转染48 h后,p65和bcl-2蛋白表达水平下调,而促凋亡蛋白bax的表达上升,进一步caspase-3/9的活性也显著升高。流式细胞术结果表明,p65siRNA能明显诱导SCL-1细胞发生凋亡,其早期凋亡的比率为20.28%±1.87%,显著高于未处理组和对照siRNA组(凋亡率分别为9.13%±1.51%和9.37%±1.38%,F=47.532,P<0.01)。结论:p65siRNA能够阻断皮肤鳞癌细胞中NF-κB信号通路,并下调抑凋亡蛋白bcl-2的表达,上调促凋亡蛋白bax的表达以及提高caspase的活性,提示NF-κB信号通路有望成为皮肤鳞癌基因治疗的分子靶点。

Objective：To evaluate the siRNA-mediated inhibitory effect of nuclear factor-kappa B（NF-κB） p65 on expression of p65,and explore the effect of blockade of NF-κB signal pathway on cell apoptosis in cutaneous squamous cell carcinoma（cutaneous SCC）.Methods： Cutaneous SCC cell line SCL-1 cells were transfected with 50 nmol/L p65 siRNA.The expression level of p65 mRNA was measured using RT-PCR method at 0,24,48 and 72 h.Expressions of p65,bcl-2 and bax proteins were determined using Western blotting.Activities of caspase-3/9 was detected by Caspase-Glo-3/7,8 and 9 kit.Finally,cell apoptosis was detected using flow cytometry.Results： The expression level of p65 mRNA in Cutaneous SCC SCL-1 cells was obviously down-regulated 48 h after transfection with p65 siRNA,and a significant difference was detected,as compared with 0 h after（0.23±0.10 vs.0.66±0.05,P〈0.05）.The protein levels of p65 and bcl-2 decreased,and the bax protein level and activities of caspase-3/9 increased after transfection with p65 siRNA at h 48.Further,the results of flow cytometry demonstrated that p65 siRNA could induce apoptosis of SCL-1 cells,and cell apoptosis ratio（20.28%±1.87%） in p65 siRNA group was significantly higher than that in the untreated group and control siRNA group（9.13%±1.51% and 9.37%±1.38%,respectively,F=47.532,P〈0.01）.Conclusion： p65 siRNA can block NF-κB signal pathway,down-regulate expression of bcl-2,elevate the bax level and increase the activities of caspase-3/9,suggesting that NF-κB signal pathway may be a key molecular target for therapy of cutaneous SCC.