U0126通过下调p-ERK1/2和GM130的表达对人胃癌细胞MKN-45的影响

Effect of U0126 on Human Gastric Cell MKN-45 by Down-Regulating the Expression of p-ERK1/2 and GM130

探讨ERK1/2通路抑制剂U0126对人胃癌MKN-45细胞增殖、凋亡、侵袭和迁移能力的影响及机制。首先用CCK-8法测定U0126对MKN-45细胞增殖的影响并筛选药物作用的最适浓度和时间用于后续实验,并将实验分为药物处理组,阴性对照组和空白对照组。然后通过流式细胞术、Transwell迁移实验和Matrigel侵袭实验分别检测细胞凋亡、迁移和侵袭能力,RT-q PCR检测各组GM130的m RNA水平的变化,Western blotting检测ERK1/2、p-ERK1/2、MMP-9、MMP-2、GM-130的蛋白水平的变化。实验发现10μmol/L、20μmol/L、30μmol/L、40μmol/L浓度的U0126均能抑制MKN-45细胞的增殖,其中20μmol/L浓度作用48 h效果最强(p<0.05),且该药物作用条件下MKN-45细胞凋亡增加,迁移和侵袭作用减弱(p<0.05)。Western blotting结果显示,药物处理组ERK1/2蛋白水平不变,而p-ERK1/2、MMP-9、MMP-2、GM-130的蛋白水平均明显低于阴性对照组和空白对照组(p<0.05)。RT-q PCR结果显示,药物处理组GM130的m RNA水平明显低于阴性对照组和空白对照组(p<0.05)。以上研究结果均表明U0126可促进MKN-45细胞凋亡,降低细胞体外增殖、迁移和侵袭的能力,其机制可能与p-ERK1/2和GM130的表达被抑制有关。

In the study,the effect and mechanism of ERK1/2 pathway inhibitor U0126 on gastric cancer cell MKN-45 proliferation,apoptosis,migration and invasion ability were investigated.At first,we used CCK-8 to determine the influence of U0126 on MKN-45 cell proliferation and screened optimal concentration and time of U0126 for follow-up studies.The experiment was divided into drug treatment group,negative control group and blank control group.Then,flow cytometry,transwell migration assay and Matrigel invasion assay were used to detect cell apoptosis,migration and invasion ability,respectively.RT-q PCR was used to analyze the GM130 m RNA level of each group.Western blotting was carried out to detect protein level of GM130,ERK1/2,p-ERK1/2,MMP-9,and MMP-2.Experiments showed that 10 μmol/L,20 μmol/L,30 μmol/L,40 μmol/L U0126 all could restrain MKN-45 cell proliferation,and 20 μmol/L for 48 h had the most powerful effect.Under this condition,MKN-45 apoptosis increased,but invasion and migration capacity was suppressed（p〈0.05）.Western blotting results showed that ERK1/2 protein level in cells treated with U0126 was invariant,but p-ERK1/2,MMP-9,MMP-2and GM130 protein level were significantly lower than those of negative control group and blank control group（p〈0.05）.Moreover,RT-q PCR showed that m RNA level of drug treatment group GM130 was significantly lower than those of negative control group and blank control group（p〈0.05）.Results above suggested that U0126 could promote MKN-45 cell apoptosis and inhibit the ability of cell proliferation,migration and invasion in vitro,and the mechanism might be related to the inhibition of the expression of p-ERK1/2 and GM130.