应激诱导磷酸化蛋白1调控分化抑制因子3表达在胃癌细胞上皮-间充质转化及侵袭迁移过程中的作用及其机制

Effects and mechanism of stress induced phosphoprotein 1 regulates ID3 expression on invasion, migration and epithelial-mesenchymal transition in gastric cancer cells

目的观察应激诱导磷酸化蛋白1(STIP1)通过调控分化抑制因子3(ID3)的表达在胃癌细胞上皮-间充质转化及侵袭迁移过程中的作用并探讨其机制。方法Westernblot和实时定量反转录聚合酶链反应(RT-qPCR)法检测胃癌细胞(BGC803)和正常胃黏膜细胞(GES-1)2株细胞中STIP1、ID3的mRNA及蛋白的表达。转染STIP1过表达载体和小干扰RNA(siRNA)感染序列至胃癌细胞,检测STIP1和ID3的表达。细胞计数试剂盒(CCK-8)法检测细胞增殖活力,Transwell检测细胞迁移及侵袭,Westernblot检测上皮型钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)的表达,并与阴性转染细胞比较。结果STIP1mRNA和蛋白在BGC803的相对表达量分别为1.851±0.058,2.033±0.107,明显高于GES-1中的1.000±0.034,1.000±0.031(t=21.924,P=0.000;t=16.061,P=0.004)。ID3mRNA和蛋白在BGC803的相对表达量分别为1.629±0.063,2.374±0.096,明显高于GES-1中的1.000±0.025,1.000±0.029(t=16.074,P=0.004;t=23.731,P=0.002)。转染STIP1过表达载体后ID3mRNA的相对表达量为2.018±0.124,较对照组1.331±0.036(t=9.216,P=0.012)明显升高;转染48h和72h后细胞增殖水平明显高于对照组(t=10.594,P=0.002;t=8.269,P=0.004);侵袭细胞数、迁移细胞数较对照组明显增加(t=5.669,P=0.011;t=5.265,P=0.013);Vimentin的表达明显升高(t=7.410,P=0.005),E-cadherin明显降低(t=-5.821,P=0.010)。转染STIP1siRNA感染序列后ID3mRNA的相对表达量为0.732±0.037,较对照组的1.252±0.051明显降低(t=-14.295,P=0.001);转染48h、72h后细胞增殖水平明显低于对照组(t=-19.851,P=0.000;t=-19.222,P=0.008);侵袭细胞数、迁移细胞数较对照组明显减少(t=-5.822,P=0.010;t=-4.859,P=0.017);Vimentin的表达明显降低(t=-6.244,P=0.008),E-cadherin明显升高(t=7.697,P=0.005)。结论STIP1调控ID3表达促进胃癌细胞增殖、侵袭、迁移和上皮-间充质转化。

Objective To investigate the effects and mechanism of stress induced phosphoprotein 1 (STIP1) on invasion, migration and Epithelial-mesenchymal transition through regulating the expression of ID3 in gastric cancer cells. Methods The expression levels of STIP1 and ID3 were detected by Western blotting and real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) in two cells (BGC803 and GES-1). To detect the change of expression of STIP1 and ID3, the STIP1 over-expression vector and small interfering RNA (siRNA) were transfected into gastric cancer cells. Cell proliferation was detected by cell counting kit-8 (CCK-8) assay, invasion and migration were detected by Transwell, E-cadherin and Vimentin were detected by Western blotting. Results The expression of STIP1 mRNA and protein in BGC803 cells were significantly higher than those in GES-1 (1.851±0.058 vs. 1.000±0.034, t=21.924, P=0.000; 2.033±0.107 vs. 1.000±0.031, t=16.061, P=0.004). The expression of ID3 mRNA and protein in BGC803 cells were significantly higher than those in GES-1 (1.629±0.063 vs. 1.000±0.025, t=16.074, P=0.004; 2.374±0.096 vs. 1.000±0.029, t=23.731, P=0.002). The results showed that ID3 mRNA expression was significantly higher (2.018±0.124 vs. 1.331±0.036, t=9.216, P=0.012), cell proliferation after transfected for 48 h and 72 h (t=10.062, P=0.002; t=6.466, P=0.008) were significantly increased, the number of invasion and migration cells were significantly increased (t=5.669, P=0.011; t=5.265, P=0.013), Vimentin expression was higher (t=7.410, P=0.005) and E-cadherin was lower (t=-5.821, P=0.010) by over-expression of STIP1. Knockdown of STIP1 decreased ID3 mRNA expression (0.732±0.037 vs. 1.252±0.051, t=-14.295, P=0.001), cell proliferation after transfected for 48 h and 72 h (t=-19.851, P=0.000; t=-19.222, P=0.008), the number of invasion and migration cells (t=-5.822, P=0.010; t=-4.859, P=0.017), Vimentin expression (t=-6.244, P=0.008) and increased E-cadherin expression (t=7.697, P=0.005). Conclusion STIP1 can increase cell proliferation, invasion, migration and Epithelial-mesenchymal transition through regulating the expression of ID3 in gastric cancer cells.