EphA2基因沉默对人食管癌EC9706细胞迁移、侵袭的影响及其机制研究

Effect and mechanism of EphA2 gene silencing on migration and invasion of human esophageal carcinoma EC9706 cells

目的探讨促红细胞生成素产生肝细胞受体A2(EphA2)基因沉默对人食管癌EC9706细胞迁移、侵袭的影响及其作用机制。方法取对数生长期人食管癌EC9706细胞随机分为对照组、空载组及沉默组,分别转染Lipofectamine;2000、含无义随机序列(siRNA-NC-PGCsi3.0)和EphA2干扰序列(EphA2-siRNA-PGCsi3.0)质粒和脂质体的复合物。稳定转染后,Brd U法检测细胞增殖能力,划痕实验检测细胞迁移能力,Transwell小室实验检测细胞侵袭能力,qRT-PCR和Western blotting分别检测细胞中EphA2、Wnt1、β-连环蛋白(β-catenin)、E-钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)mRNA及蛋白相对表达量。结果对照组、空载组、沉默组BrdU阳性率分别为(26.48±2.79)%、(23.52±2.57)%、(13.29±2.06)%,Brd U阳性率组间比较,差异有统计学意义(F=38.560,P=0.000)。与对照组、空载组比较,沉默组Brd U阳性率降低(P <0.05);对照组与空载组比较,差异无统计学意义(P>0.05)。对照组、空载组、沉默组细胞划痕愈合率分别为(83.16±8.31)%、(82.35±7.24)%、(34.24±5.27)%,细胞划痕愈合率组间比较,差异有统计学意义(F=78.869,P=0.000)。与对照组、空载组比较,沉默组细胞划痕愈合率降低(P <0.05);对照组与空载组比较,差异无统计学意义(P>0.05)。对照组、空载组、沉默组穿膜细胞数分别为(326.74±33.15)个、(331.27±34.59)个、(126.23±26.18)个,穿膜细胞数组间比较,差异有统计学意义(F=68.997,P=0.000),与对照组、空载组比较,沉默组穿膜细胞数减少(P <0.05);对照组与空载组比较,差异无统计学意义(P>0.05)。细胞中EphA2、Wnt1、β-catenin、Ecadherin、Vimentin mRNA相对表达量比较,差异有统计学意义(P <0.05),与对照组、空载组比较,沉默组Wnt1、β-catenin、Vimentin mRNA相对表达量降低(P <0.05),E-cadherin mRNA相对表达量升高(P <0.05)。对照组、空载组、沉默组细胞中EphA2、Wnt1、β-catenin、E-cadherin、Vimentin蛋白相对表达量比较,差异有统计学意义(P <0.05),与对照组、空载组比较,沉默组EphA2、Wnt1、β-catenin、Vimentin蛋白相对表达量降低(P <0.05),E-cadherin蛋白相对表达量升高(P <0.05)。对照组和空载组上述指标比较,差异无统计学意义(P>0.05)。结论 EphA2基因沉默对人食管癌EC9706细胞迁移和侵袭有一定的抑制作用,其机制可能与阻止Wnt/β-catenin信号通路相关。

Objective To investigate the effect and mechanism of erythropoietin producing hepatoma receptor(Eph) receptor A2 gene silencing on migration and invasion of human esophageal cancer EC9706 cells.Methods Human esophageal cancer EC9706 cells in logarithmic growth phase were randomly divided into control group, empty group, and silent group, transfected with Lipofectamine;2000, containing nonsense random sequence(siRNA-NC-PGCsi3.0) and EphA2 interference sequence(EphA2-siRNA-PGCsi3.0). After stable transfection, BrdU method was used to detect cell proliferation ability, scratch test to detect cell migration ability, Transwell cell test to detect invasion ability, qRT-PCR and Western blotting to detect the mRNA and protein expression levels of EphA2, Wnt1, β-catenin, E-cadherin, Vimentin in cells. Results The relative expression of EphA2 mRNA in the control group, no-load group, and silent group were(1.78 ± 0.17),(1.82 ± 0.18),(0.37 ± 0.11), respectively. The difference between the groups was statistically significant(P < 0.05). Compared with the control group and the noload group, the relative expression of EphA2 m RNA in the silent group decreased(P < 0.05). The positive rates of Brd U in the control group, the no-load group, and the silent group were(26.48 ± 2.79) %,(23.52 ± 2.57) %,(13.29 ±2.06) %, respectively. The difference between the groups was statistically significant(P < 0.05). Compared with the control group and the no-load group, the positive rate of Brd U in the silent group was lower(P < 0.05). The cell scratch healing rates of the control group, no-load group, and silent group were(83.16 ± 8.31) %,(82.35 ±7.24) %,(34.24 ± 5.27) %, respectively. The difference between the groups was statistically significant(P < 0.05). Compared with the control group and the no-load group, the cell scratch healing rate of the silent group increased(P < 0.05).The number of transmembrane cells in the control group, the no-load group, and the silent group were(326.74 ±33.15),(331.27 ± 34.59),(126.23 ± 26.18), respectively. The difference between the groups was statistically significant(P < 0.05). Compared with the control group and the no-load group, the number of transmembrane cells in the silent group decreased(P < 0.05). The differences of relative expression levels of Wnt1, β-catenin, E-cadherin,and Vimentin mRNA were statistically significant(P < 0.05). Compared with the control group and the no-load group, the relative expression of Wnt1, β-catenin, Vimentin m RNA in the silent group decreased, and the relative expression of E-cadherin m RNA increased(P < 0.05). The difference of relative expression levels of EphA2, Wnt1,β-catenin, E-cadherin, and Vimentin protein in cells were statistically significant(P < 0.05). Compared with the control group and the no-load group, the relative expression of EphA2, Wnt1, β-catenin, and Vimentin protein in the silent group decreased, and the relative expression of E-cadherin protein increased(P < 0.05). The comparison of the above indicators between the control group and the no-load group showed no significant difference(P > 0.05).Conclusion EphA2 gene silencing has a certain inhibitory effect on the migration and invasion of human esophageal cancer EC9706 cells, and its mechanism may be related to preventing the Wnt/β-catenin signaling pathway.