摘要
目的:观察biglycan及血管内皮生长因子(vascular endothelial growth factor,VEGF)对结肠癌细胞系HCT116迁移、侵袭能力的影响并探讨其可能的机制。方法:在稳定转染biglycan的结肠癌细胞系HCT116中转染VEGF siRNA,实验设置未转染对照组(mock组)、空载质粒和非特异性干扰片段转染对照组(vector+siRNA-NC组)、biglycan cDNA和非特异性干扰片段转染组(biglycan+siRNA-NC组)以及biglycan cDNA和VEGF siRNA共转染组(biglycan+siRNA-VEGF组)。采用实时荧光定量逆转录聚合酶链反应(RT-PCR)法检测biglycan和VEGF的mRNA表达;采用划痕实验检测细胞的迁移能力;采用Transwell法检测细胞的侵袭能力;采用蛋白质印迹(Western blotting)法检测SNAIL、E-钙黏蛋白(E-cadherin)、波形蛋白(vimentin)、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶-9(MMP-9)的表达;采用明胶酶谱法检测MMP-2、MMP-9的活性。结果:与mock组及vector+siRNA-NC组比较,biglycan+siRNA-NC组细胞的迁移、侵袭能力均显著提高(P<0.05),vimentin、MMP-2、MMP-9、SNAIL蛋白的表达水平显著提高,E-cadherin蛋白的表达水平则显著下降(P<0.05),MMP-2和MMP-9的活性显著提高(P<0.05)。与biglycan+siRNA-NC组比较,biglycan+siRNA-VEGF组细胞的迁移、侵袭能力均显著降低(P<0.05),SNAIL、vimentin、MMP-2、MMP-9蛋白的表达水平均显著下降,E-cadherin蛋白的表达水平显著提高(P<0.05),MMP-2、MMP-9的活性显著下降(P<0.05)。结论:biglycan通过促进VEGF的表达来促进结肠癌细胞系HCT116的迁移和侵袭,下调结肠癌细胞中VEGF的表达可逆转biglycan对HCT116迁移和侵袭的促进作用。
Objective：To observe the effect of biglycan and vascular endothelial growth factor（VEGF） on the migration and invasion of HCT116 cell line in vitro and to investigate the possible mechanism.Methods：VEGF siRNA was transfected into HCT116 cell line which had been stably transfected with biglycan.Untransfected control group（mock group）,control group transfected with empty plasmid and non-specific interference fragment （vector ＋ siRNA-NC group）,group transfected with biglycan cDNA and non-specific interference fragment （biglycan ＋ siRNA-NC group）,group co-transfected with biglycan cDNA and VEGF siRNA（biglycan ＋ siRNA-VEGF group） were set.The expressions of biglycan and VEGF mRNA were detected by real-time fluorescent quantitative reverse transcription-polymerase chain reaction （RT-PCR）.The migration was detected by wound healing assay and the invasion was detected by Transwell assay.The expressions of SNAIL,E-cadherin,vimentin,MMP-2,MMP-9 proteins were detected by Western blotting.The activity of MMP-2 and MMP-9 were detected by gelatin zymography assay.Results：Compared with the mock group and vector ＋ siRNA-NC group,the migrational and invasive ability of cells in biglycan ＋ siRNA-NC group significantly increased （P 〈 0.05）; the expressions of vimentin,MMP-2,MMP-9 and SNAIL proteins increased significantly,while that of E-cadherin decreased（P〈0.05） ; the activity of MMP-2 and MMP-9 significantly increased（P〈0.05）.Compared with the biglycan ＋ siRNA-NC group,the migrational and invasive ability of cells in biglycan ＋ siRNA-VEGF group significantly decreased（P〈0.05）; the expressions of SNAIL,vimentin,MMP-2 and MMP-9 proteins significantly decreased,while that of E-cadherin significantly increased （P〈0.05）,and the activity of MMP-2 and MMP-9 decreased（P〈0.05）. Conclusions： biglycan promotes the migration and invasion of colon cancer cells HCT116 by increasing the expression of VEGF,and the effects by biglycan on colon cancer can be reverse
出处
《中国临床医学》
2014年第4期391-396,共6页
Chinese Journal of Clinical Medicine
基金
国家自然科学基金项目(编号:81201968)
辽宁省自然科学基金项目(编号:201102111)
辽宁省科技攻关项目(编号:2012225016)
