血小板衍生生长因子D通过ERK信号通路对肺癌H1299细胞增殖、迁移和侵袭的影响及其机制

Effects of platelet-derived growth factor D on proliferation,migration and invasion of lung cancer H1299 cells through ERK signaling pathway and their mechanisms

目的:探讨血小板衍生生长因子D(PDGF-D)对人肺癌H1299细胞增殖、迁移和侵袭的影响,并阐明其可能的作用机制。方法:选用人肺癌H1299细胞,分为对照组(转染空载体)和PDGF-D组(转染GV230-PDGF-D质粒),同时设空白组,采用实时荧光定量PCR(RT-qPCR)法和Western blotting法检测各组细胞中PDGF-D mRNA表达水平和蛋白表达量。H1299细胞分为对照组(转染空载体)、PDGF-D组(转染GV230-PDGF-D质粒)、PDGF-D+PD98059组(转染GV230-PDGF-D质粒+ERK抑制剂PD98059)和PD98059组,PD98059终浓度为10μmol·L^(-1)。MTT法检测各组细胞增殖活性,划痕实验检测各组细胞迁移率,Transwell实验检测各组细胞中侵袭细胞数,Western blotting法检测各组细胞中磷酸化细胞外信号调节基酶(p-ERK)、细胞外信号调节基酶(ERK)、锌指转录因子Snail、基质金属蛋白酶1(MMP-1)和跨膜黏附糖蛋白CD44的蛋白表达水平。结果:RT-qPCR法检测,与空白组和对照组比较,PDGF-D组细胞中PDGF-D mRNA表达水平明显升高(P<0.01);Western blotting法检测,空白组和对照组细胞中无PDGF-D蛋白表达,与空白组和对照组比较,PDGF-D组细胞中PDGF-D蛋白表达量明显增加。与对照组比较,PDGF-D组细胞增殖活性和细胞迁移率明显升高(P<0.05或P<0.01),侵袭细胞数明显增加(P<0.01),细胞中p-ERK、Snail、MMP-1和CD44蛋白表达水平明显升高(P<0.05);与PDGF-D组比较,PDGF-D+PD98059组细胞增殖活性和细胞迁移率明显降低(P<0.05或P<0.01),侵袭细胞数明显减少(P<0.01),细胞中p-ERK、Snail、MMP-1和CD44蛋白表达水平明显降低(P<0.05);与PDGF-D+PD98059组比较,PD98059组细胞增殖活性和细胞迁移率明显降低(P<0.05或P<0.01),侵袭细胞数明显减少(P<0.01),细胞中p-ERK、Snail、MMP-1和CD44蛋白表达水平降低(P<0.05或P<0.01)。结论:PDGF-D可促进人肺癌H1299细胞增殖、迁移和侵袭,其机制可能与其通过ERK信号通路上调Snail、MMP-1和CD44的蛋白表达有关。

Objective:To investigate the effects of platelet-derived growth factor D(PDGF-D)on the proliferation,migration and invasion of lung cancer H1299 cells,and to elucidate their possible mechanisms.Methods:The lung cancer H1299 cells were divided into control group(transfected with empty plasmid)and PDGF-D group(transfected with GV230-PDGF-D plasmid);meanwhile,blank group was set up.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods were used to detect the expression levels of PDGF-D mRNA and the expression amounts of PDGF-D protein in the cells in various groups.The H1299 cells were divided into control group(transfected with empty vector),PDGF-D group(transfected with GV230-PDGF-D plasmid),PDGF-D+PD98059(transfected with GV230-PDGF-D plasmid and treated with PD98059,an inhibitor of ERK),and PD98059 group;the final concentration of PD98059 was 10μmol·L^(-1).MTT assay was used to detect the proliferation activities of cells in various groups.The migration rates of cells in various groups were detected by scratch test.The number of invasion cells in various groups was detected by Transwell assay.The expression levels of phosphorylated signal extracellular regulatory enzyme(p-ERK),extracellular signal regulatory enzyme(ERK),zinc finger transcription factor Snail,matrix metalloproteinases-1(MMP-1)and transmembrane adhesion glycoproteins CD44 proteins in various groups were detected by Western blotting method.Results:The results of RT-qPCR method showed that compared with blank group and control group,the expression levels of PDGF-D mRNA in the cells in PDGF-D group were significantly increased(P<0.01).The Western blotting results showed that there was no PDGF-D protein expression in the cells in blank group and control group;compared with blank group and control group,the expression amount of PDGF-D protein in PDGF-D group was siginificantly increased.Compared with control group,the proliferation activity of the cells and the migration rate in PDGF-D were significantly increased(P<0.05 or P<0.01),the number of invasion cells was significantly increased(P<0.01),and the expression levels of p-ERK,Snail,MMP-1 and CD44 proteins in the cells were increased(P<0.05).Compared with PDGF-D group,the proliferation activity of the cells and the migration rate in PDGF-D+PD98059 group were significantly decreased(P<0.05),the number of invasion cells was decreased(P<0.05),and the expression levels of p-ERK,Snail,MMP-1 and CD44 proteins in the cells were decreased(P<0.05).Compared with PDGF-D+PD98059 group,the proliferation activity of the cells and the migration rate in PD98059 group were significantly decreased(P<0.05 or P<0.01),the number of invasion cells was decreased(P<0.01),and the expression levels of p-ERK,Snail,MMP-1 and CD44 proteins in the cells were decreased(P<0.05 or P<0.01).Conclusion:PDGF-D can promote the proliferation,migration and invasion of lung cancer H1299 cells,and their mechanisms may be related to the up-regulation of Snail,MMP-1 and CD44 protein expressions through ERK signaling pathway.