ERK-VEGF/MMP-9信号通路与胃癌细胞增殖、转移和侵袭的关联机制

Association of ERK-VEGF/MMP-9 signaling pathway with proliferation,metastasis and invasion of gastric cancer cells

目的探讨ERK-VEGF/MMP-9信号通路与胃癌细胞增殖和预后转归关联性。方法离体培养人胃癌细胞MGC-803,72个培养皿的细胞分为三组:对照组(n=24);阻断组(n=24);转染组(n=24)。对照组细胞不做任何干预;阻断组细胞应用特异性ERK-VEGF/MMP-9信号通路抑制剂GDC-0994对胃癌MGC-803细胞的ERK-VEGF/MMP-9信号通路进行抑制;转染组细胞转染ERK1质粒。用MTT法检测胃癌MGC-803细胞生长,采取流式细胞仪检测胃癌MGC-803细胞周期及凋亡的变化;采用细胞迁移和侵袭实验分析ERK-VEGF/MMP-9信号通路对胃癌细胞侵袭能力的影响;采取RT-PCR法检测ERK、VEGF以及MMP-9 mRNA,用Westem blot法检测ERK、VEGF以及MMP-9蛋白表达。结果阻断组MGC-803细胞增殖低于对照组和转染组,转染质粒ERK1则可提高MGC-803细胞增殖率(P<0.05)。阻断组凋亡数量(20.59±3.27)高于对照组(F=281.588,P<0.05)。在G0/G1期,阻断组细胞凋亡数量与对照组比较,并无显著差异(P>0.05),在S期和G2/M期,阻断组细胞凋亡比率高于对照组(P<0.05)。转染质粒ERK1后,其凋亡数量低于对照组(P<0.05)。在G0/G1期,阻断组细胞凋亡数量与对照组比较,无显著差异(P>0.05),而在S期和G2/M期,阻断组细胞凋亡比率低于对照组(P<0.05)。与对照组(314.52±52.11)比较,阻断组MGC-803(185.42±30.2)细胞迁移数量降低,转染质粒ERK1后MGC-803细胞迁移数量增多(P均<0.05)。与对照组比较,阻断组MGC-803细胞侵袭降低,转染质粒ERK1后MGC-803细胞侵袭能力增强(P<0.05)。阻断组,MGC-803细胞中信号通路的关键效应蛋白ERK、VEGF、MMP-9蛋白表达水平较对照组下调;转染质粒ERK1后ERK、VEGF、MMP-9 mRNA蛋白表达水平较对照组上调。结论ERK-VEGF/MMP-9信号通路若受到拮抗,则可抑制胃癌肿瘤细胞的增殖并诱导细胞产生凋亡,并下调胃癌细胞迁移和侵袭能力。

Objective To investigate the relationship between ERK-VEGF/MMP-9 signaling pathway and proliferation and prognosis of gastric cancer cells.Methods Human gastric cancer cell line MGC-803 was cultured in vitro.72 dishes were divided into three groups:control group(n=24),blocking group(n=24),transfection group(n=24).The control group did not receive any intervention.Specific ERK-VEGF/MMP-9 signaling pathway inhibitor GDC-0994 was used to inhibit ERK-VEGF/MMP-9 signaling pathway in gastric cancer MGC-803 cells in the blocking group.ERK1 plasmid was transfected into the transfected cells.The growth of gastric cancer MGC-803 cells was detected by MTT assay.The cell cycle and apoptosis of gastric cancer MGC-803 were detected by flow cytometry.The effects of ERK-VEGF/MMP-9 signaling pathway on the invasive ability of gastric cancer cells were analyzed by cell migration and invasion experiments.ERK,VEGF and MMP-9 were detected by RT-PCR.The expressions of ERK,VEGF and MMP-9 were detected by Westem blot.Results The proliferation of MGC-803 cells in the blocking group was lower than that in the control group and the transfection group.The transfection plasmid ERK1 could increase the proliferation rate of MGC-803 cells(F=296.284,P<0.05).The number of apoptotic cells in the blocking group(20.59+3.27)was higher than that in the control group(F=281.588,P<0.05).In G0/G1 phase,there was no significant difference in the number of apoptotic cells between the blocking group and the control group(P>0.05).In S phase and G2/M phase,the percentage of apoptotic cells in the blocking group was higher than that in the control group(P<0.05).After transfection of plasmid ERK1,the number of apoptotic cells was lower than that of control group(P<0.05).In G0/G1 phase,there was no significant difference in the number of apoptotic cells between the blocking group and the control group(P>0.05).In S phase and G2/M phase,the percentage of apoptotic cells in the blocking group was lower than that in the control group(P<0.05).Compared with the control group(314.52+52.11),the number of MGC-803(185.42+30.2)cells in the blocking group decreased,and MG-803(185.42+30.2)cells transfected with plasmid ERK1 decreased.The number of C-803 cells migrated increased with statistical difference(F=131.239,P<0.05).Compared with the control group,the invasion of MGC-803 cells decreased in the blocking group,and the invasion of MGC-803 cells increased after transfection of plasmid ERK1(P<0.05).In the blocking group,the expression levels of ERK,VEGF and MMP-9,the key effector proteins of signal pathway in MGC-803 cells,were down-regulated compared with the control group,and the expression levels of ERK,VEGF and MMP-9 were up-regulated after transfection of plasmid ERK1.Conclusion If ERK-VEGF MMP-9 signaling pathway is antagonized,it can inhibit the proliferation of gastric cancer cells and induce cell apoptosis,and down-regulate the migration and invasion of gastric cancer cells.