基于NF-κB信号通路探究木犀草素对宫颈癌细胞增殖和凋亡的调控作用

Study on the Regulation of Luteolin on the Proliferation and Apoptosis of Cervical Cancer Cells Based on NF-κB Signal Pathway

目的:探讨木犀草素对宫颈癌细胞增殖和凋亡的影响及其对核转录因子-κB(NF-κB)信号通路的调控作用。方法:将HeLa细胞分为对照组、5μmol/L木犀草素组、10μmol/L木犀草素组、20μmol/L木犀草素组和抑制剂组。5、10、20μmol/L木犀草素组HeLa细胞分别经不同浓度(5、10和20μmol/L)木犀草素处理24 h,对照组HeLa细胞不做处理。筛选出10μmol/L为木犀草素最适浓度,然后将细胞分为对照组、10μmol/L木犀草素组和抑制剂组[10μmol/L木犀草素+2.5μmol/L BAY 11-7082(NF-κB信号通路抑制剂)]。采用5-乙炔基-2'脱氧尿嘧啶核苷(EdU)测定细胞增殖情况,平板克隆测定细胞克隆情况,Hoechst 33258测定细胞凋亡情况,实时荧光定量PCR(RT-qPCR测定半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)mRNA和细胞周期素D1(Cyclin D1)mRNA表达,蛋白免疫印迹(Western blotting)法测定Caspase-3、Cyclin D1、NF-κB和p-NF-κB蛋白表达。结果:10、20μmol/L木犀草素组细胞增殖率、克隆形成率、Cyclin D1 mRNA和Cyclin D1蛋白相对表达量低于对照组,差异均有统计学意义(P<0.05);10、20μmol/L木犀草素组细胞凋亡数、Caspase-3 mRNA和Caspase-3蛋白相对表达量均高于对照组,差异均有统计学意义(P<0.05)。因此本实验选择低浓度(10μmol/L)木犀草素加入NF-κB通路抑制剂作为抑制剂组进行后续验证实验。抑制剂组细胞增殖率和克隆形成率低于10μmol/L木犀草素组,差异有统计学意义(P<0.05)。10μmol/L木犀草素组Cyclin D1 mRNA、Cyclin D1蛋白、p-NF-κB蛋白相对表达量低于对照组,Caspase-3 mRNA和Caspase-3蛋白相对表达量高于对照组,差异均有统计学意义(P<0.05);抑制剂组Cyclin D1 mRNA、Cyclin D1蛋白、p-NF-κB蛋白相对表达量低于10μmol/L木犀草素组,Caspase-3 mRNA和Caspase-3蛋白相对表达量高于10μmol/L木犀草素组,差异均有统计学意义(P<0.05)。结论:木犀草素可能通过抑制NF-κB信号通路诱导HeLa细胞的凋亡并减弱其增殖能力。

Objective:To investigate the effects of luteolin on the proliferation and apoptosis of cervical cancer cells and its regulation on nuclear factor-κB(NF-κB)signaling pathway.Methods:HeLa cells were divided into control group,5μmol/L luteolin group,10μmol/L luteolin group,20μmol/L luteolin group and inhibitor group.HeLa cells in 5,10 and 20μmol/L luteolin groups were treated with different concentrations(5,10 and 20μmol/L)of luteolin for 24 h,respectively,while HeLa cells in control group were not treated.10μmol/L was selected as the optimal concentration of luteolin,and then the cells were divided into control group,10μmol/L luteolin group and inhibitor group[10μmol/L luteolin+2.5μmol/L BAY11-7082(inhibitor of NF-κB signaling pathway)].Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine(EdU),cell cloning was measured by colony formation,and cell apoptosis was measured by Hoechst 33258 stainig.Real-time fluorescent quantitative PCR(RT-qPCR)was used to measure the expression of Caspase-3 and Cyclin D1 mRNA.The protein expressions of Caspase-3,Cyclin D1,NF-κB and p-NF-κB were determined by Western blotting.Results:The cell proliferation rate,clone formation rate,relative expression of Cyclin D1 mRNA and protein in 10 and 20μmol/L luteolin groups were lower than those in the control group,and the differences were statistically significant(P<0.05).The number of apoptosis,mRNA and protein expression of Caspase-3 in 10 and 20μmol/L luteolin groups were higher than those in the control group,and the differences were statistically significant(P<0.05).Therefore,in this experiment,a low concentration of 10μmol/L luteolin was added to the NF-κB pathway inhibitor as the inhibitor group for subsequent verification experiments.The cell proliferation rate and colony formation rate of the inhibitor group were lower than those of the 10μmol/L luteolin group,and the difference was statistically significant(P<0.05).The relative expressions of Cyclin D1 mRNA,Cyclin D1 protein and p-NF-κB protein in 10μmol/L luteolin group were lower than those in the control group,and the relative expressions of Caspase-3 mRNA and Caspase-3 protein were higher than those in the control group,and the differences were statistically significant(P<0.05).The relative expression levels of Cyclin D1 mRNA,Cyclin D1 protein and p-NF-κB protein in the inhibitor group were lower than those in the 10μmol/L luteolin group,and the relative expression levels of Caspase-3 mRNA and Caspase-3 protein were higher than those in the 10μmol/L luteolin group.The difference was statistically significant(P<0.05).Conclusion:Luteolin may induce apoptosis and decrease proliferation of HeLa cells by inhibiting NF-κB signaling pathway.