TLR9的激活对食管鳞癌细胞的增殖侵袭能力及NF-κB表达的影响

The effects of Toll-like receptor 9 activation on the proliferation,invasion and expression of NF-κB of esophageal cancer cell

目的探索Toll样受体9(Toll-like receptor 9,TLR9)的激活对食管鳞癌细胞的增殖侵袭能力及NF-κB表达的影响。方法流式细胞术检测TLR9在食管鳞癌细胞TE-3、TE-13、TE-15、TE-30中的表达;免疫荧光法检测TLR9在TE-13细胞中的表达;用CpG寡脱氧核苷酸(CpG-ODN)激活TE-13,实验分为阴性对照组(control)、低剂量CpG-ODN组(1μg/mL)、中剂量CpG-ODN组(3μg/mL)和高剂量CpG-ODN组(12μg/mL)。RT-qPCR检测食管鳞癌细胞TE13中TLR9和NF-κB mRNA相对表达量的变化;采用CCK-8和Transwell法分别检测TLR9的激活对TE-13细胞增殖和侵袭能力的影响。结果①TLR9在TE-13表达量高于其他3种食管鳞癌细胞系。②3、12μg/mL CpGODN作用后TE-13细胞中TLR9被充分激活并且上调NF-κB的表达,TLR9和NF-κB的表达显著均高于阴性对照组(P<0.05);低剂量CpG-ODN组与阴性组相比差异无统计学意义(P>0.05)。③CCK-8和Transwell法检测发现CpGODN浓度在3、12μg/mL时TLR9的激活可显著增加TE-13细胞的增殖和侵袭能力(P<0.05),激活作用具有浓度依赖性。低剂量CpG-ODN组与阴性组相比差异无统计学意义(P>0.05)。结论CpG-ODN可通过TLR9/NF-κB促进食管鳞癌细胞的增殖和侵袭,提示TLR9可能是治疗食管鳞癌的重要靶点。

ObjectiveTo investigate the effect of TLR9 activation on proliferation,invasion,and the expression ofNF-κB of esophageal cancer cell.MethodsThe expression of TLR9 protein in esophageal cancer cell TE-3,TE-13,TE-15,TE-30 was detected by flow cytometry.The expression of TLR9 in TE-13 was detected by immunefluorescence staining.Theexperiment that treating TE-13 with Cp G-ODN was divided into four groups:(1)negative control group(control);(2)low doseCp G-ODN group(1μg/m L);(3)medium dose Cp G-ODN group(3μg/m L).(3)High dose Cp G-ODN group(12μg/m L).The expression of TLR9 m RNA and NF-κB m RNA in TE-13 was detected by real-time fluorescent quantitative PCR.Thechange of proliferation and invasion activity was separately detected by CCK-8 and Transwell assay.Results(1)Theexpression of TLR9 in TE-13 was higher than others.(2)Compared with the negative control group,the expression of TLR9 m RNA and NF-κB m RNA in TE-13 cells cultured with 3,12μg/m L Cp G-ODN was much higher(P<0.05);When treatingTE-13 with 1μg/m L Cp G-ODN,the difference between two group had no statistically significant(P>0.05).(3)The activityof proliferation and invasion of TE-13 cells cultured by 3,12μg/m L Cp G-ODN was significantly higher than negative controlgroup with the TLR9 activation in a dose-dependent manner(P<0.05);Low dose Cp G-ODN group had no difference withnegative control group(P>0.05).ConclusionCp G-ODN can enhance the proliferation and invasion activity of TE-13 cellsby activating TLR9/NF-κB signaling pathway.Thus,TLR9/NF-κB signaling pathway might be the new target for esophagealcancer treatment.