木犀草素调控食管癌细胞生物学行为及其机制

Luteolin regulates the biological behavior of esophageal cancer cells and its mechanism

目的探讨木犀草素对食管癌细胞生物学行为的影响及其机制。方法分别用0 μmol/L(对照组)、25 μmol/L(低剂量木犀草素组)、50 μmol/L(中剂量木犀草素组)及100 μmol/L(高剂量木犀草素)浓度木犀草素处理EC9706细胞3 d。分别转染anti-微小核糖核酸(miR)-NC、anti-miR-718、miR-NC及miR-718 mimics至EC9706细胞, 转染anti-miR-NC及anti-miR-718细胞分别标记为anti-miR-NC组及anti-miR-718组。用100 μmol/L浓度木犀草素孵育转染miR-NC(高剂量木犀草素+miR-NC组)及转染miR-718 mimics(高剂量木犀草素+miR-718组)至EC9706细胞。溴化-3-(4, 5-二甲基-2-噻唑)-2, 5-二苯基四氮唑(MTT)检测细胞增殖;Transwell检测细胞侵袭及迁移;流式细胞仪检测细胞凋亡;实时荧光定量聚合酶链反应(RT-qPCR)检测miR-718表达水平;蛋白质印迹法检测蛋白表达。两组间比较行t检验, 多组间比较采用单因素方差分析, 组间两两比较采用SNK检验。结果低、中、高剂量木犀草素组细胞增殖活力显著低于对照组(分别1.34±0.06、0.97±0.05、0.54±0.04比1.53±0.08), 差异有统计学意义(F=53.311, P<0.05)。低、中、高剂量木犀草素组迁移细胞数、侵袭细胞数、细胞周期蛋白D1(CyclinD1)、基质金属蛋白酶2(MMP)-2、MMP-9及B细胞淋巴瘤-2(bcl-2)表达显著低于对照组[迁移细胞数分别为(116.73±5.09)、(87.74±3.14)、(51.46±3.46)个比(136.67±6.13)个, 侵袭细胞数分别为(103.29±4.45)、(65.17±2.92)、(29.34±1.16)个比(121.48±5.89)个, Cyclin D1分别为0.71±0.04、0.49±0.03、0.24±0.02比0.79±0.05, MMP-2分别为0.66±0.04、0.49±0.03、0.31±0.02比0.75±0.05, MMP-9分别为0.57±0.04、0.41±0.02、0.23±0.02比0.69±0.04, bcl-2分别为0.61±0.03、0.46±0.03、0.25±0.02比0.69±0.05], 凋亡率、p21及B细胞淋巴瘤-2相关X蛋白(bax)表达显著高于对照组[凋亡率分别为(7.89±0.92)%、(15.82±1.19)%、(24.61±1.56)%比(5.83±0.51)%, p21分别为0.42±0.03、0.59±0.04、0.83±0.05比0.31±0.02, bax分别为(0.29±0.03、0.48±0.03、0.71±0.06比0.19±0.02), 差异有统计学意义(F=64.295、104.645、58.793、44.683、35.804、30.089、35.530、32.702、38.778, P<0.05)]。低、中、高剂量木犀草素组细胞miR-718表达显著低于对照组(分别为0.89±0.05、0.67±0.04、0.41±0.03比1.01±0.07), 差异有统计学意义(F=26.443, P<0.05)。高剂量木犀草素+miR-718组细胞迁移细胞数、侵袭细胞数、Cyclin D1、MMP-2、MMP-9、bcl-2表达显著高于高剂量木犀草素+miR-NC组, 凋亡率、p21及bax表达显著低于anti-miR-NC组, 差异有统计学意义(t=8.172、14.002、7.023、10.201、6.330、7.093、9.799、6.988、9.381, P<0.05)。结论木犀草素可通过下调miR-718表达而抑制食管癌EC9706细胞的增殖、迁移和侵袭并诱导细胞凋亡。

Objective To investigate the effect of luteolin on the biological behavior of esophageal cancer cells and its mechanism.Methods EC9706 cells were treated with luteolin at concentrations of 0(control group),25 mol/L(low-dose luteolin group),50(medium-dose luteolin group)and 100μmol/L(high-dose luteolin group)for 3 d.EC9706 cells were transfected with anti-microRNA(miR)-NC,anti-miR-718,miR-NC and miR-718 mimics,respectively.The transfected miR-NCs(high-dose luteolin+miR-NC group)and miR-718 mimics(high-dose luteolin+miR-718 group)were incubated with 100μmol/L luteolin to EC9706 cells.Bromide-3-(4,5-Dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide(MTT)were used to detect cell proliferation.Transwell assay was used to detect cell invasion and migration.Flow cytometry was used to detect cell apoptosis.The real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect miR-718 expression.The protein expression was detected by Western blotting.The t test was used for comparison between two groups,one-way analysis of variance was used for comparison between multiple groups,and SNK test was used for pairwise comparison between groups.Results The number of migrating cells,the number of invasive cells,the expression levels of Cyclin D1,matrix metalloproteinases-2(MMP)-2,MMP-9 and B-cell lymphoma 2(bcl-2)in the low-,medium-and high-dose luteolin groups were significantly reduced as compared with those in the control group(for number of migrating cells:116.73±5.09,87.74±3.14,51.46±3.46 vs.136.67±6.13;for number of invasive cells:103.29±4.45,65.17±2.92,29.34±1.16 vs.121.48±5.89;for Cyclin D1:0.71±0.04,0.49±0.03,0.24±0.02 vs.0.79±0.05;for MMP-2:0.66±0.04,0.49±0.03,0.31±0.02 vs.0.75±0.05;for MMP-9:0.57±0.04,0.41±0.02,0.23±0.02 vs.0.69±0.04;for bcl-2:0.61±0.03,0.46±0.03,0.25±0.02 vs.0.69±0.05),while the apoptosis rate,p21 and B-cell lymphoma 2 associated X protein(bax)expression were significantly increased as compared with those in the control group[for apoptosis rates:(7.89±0.92)%,(15.82±1.19)%,(24.61±1.56)%vs.(5.83±0.51)%;for p21:0.42±0.03,0.59±0.04,0.83±0.05 vs.0.31±0.02;for bax:0.29±0.03,0.48±0.03,0.71±0.06 vs.0.19±0.02].The differences were statistically significant(F=64.295,104.645,58.793,44.683,35.804,30.089,35.530,32.702,38.778,P<0.05).The expression levels of miR-718 in low-,medium-and high-dose luteolin groups were significantly lower than those in the control group(0.89±0.05,0.67±0.04,0.41±0.03 vs.1.01±0.07,respectively),and the difference was statistically significant(F=26.443,P<0.05).The number of migrating cells,the number of invasive cells,the expression of Cyclin D1,MMP-2,MMP-9 and bcl-2 in the high-dose luteolin+miR-718 group were significantly increased as compared with those in the high-dose luteolin+miR-NC group.The apoptosis rate,p21 and bax expression were significantly lower than those in the anti-miR-NC group,and the difference was statistically significant(t=8.172,14.002,7.023,10.201,6.330,7.093,9.799,6.988,9.381,P<0.05).Conclusion Luteolin can inhibit the proliferation,migration and invasion of EC9706 cells and induce apoptosis by downregulating the expression of miR-718.