GRHL2对结肠癌失巢凋亡和侵袭迁移的作用研究  

Research on the effect of GRHL2on migration,invasion and anoikis of colon cancer

作　　者：汪锦江 李佳曦 邓周峰 刘子梅 乔光磊[1] 马俐君[1] WANG Jin-jiang;LI Jia-xi;DENG Zhou-feng(Department of Oncology Tongren Hospital ShanghaiJiao Tong University Schoolof Medicine,Shanghai 200336,China)

机构地区：[1]上海交通大学医学院附属同仁医院肿瘤科,上海200336

出　　处：《中国实验诊断学》2022年第6期899-904,共6页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金(81672335);上海市卫生和计划生育委员会科研课题(20174Y0231)。

摘　　要：目的探索沉默及回补粒状头样(GRHL)2基因后对人结肠癌HCT116细胞失巢凋亡和侵袭迁移功能的作用。方法应用本课题组前期构建的GRHL2基因沉默及基因回补表达的结肠癌HCT116细胞株作为实验研究组,设为基因沉默组(GRHL2-KO)和基因回补组(GRHL2-OE),以结肠癌HCT116作为空白对照组(Control),采用qRT-PCR(Quantitative Real-Time Polymerase Chain Reaction)和蛋白印迹技术(western blot)检测各组GRHL2的mRNA和蛋白表达;通过Calcein AM/EthD-1荧光双染法检测细胞失巢凋亡功能;应用划痕实验和侵袭迁移实验检测细胞迁移功能;Western blotting检测检测E-cadherin、N-cadherin和RAB25蛋白的表达。结果与空白对照组相比,基因沉默组的mRNA和蛋白基本没有表达(P<0.0001),基因回补组与基因沉默组相比mRNA和蛋白表达明显上升(P<0.0001);失巢凋亡实验结果显示,与空白对照组相比,GRHL2沉默后,存活细胞率明显增加[Calcein AM:(147.25±20.06)%vs(343.50±6.62)%,P<0.001],失巢凋亡率明显减少[EthD-1:(72.12±9.19)%vs(53.96±5.35)%,P<0.01];GRHL2回补后存活细胞率明显下降[Calcein AM:(343.50±6.62)%vs(119.73±14.48)%,P<0.0001],细胞失巢凋亡率明显回升[EthD-1:(2.25±0.10)%vs(53.96±5.35)%,P<0.001];划痕实验和侵袭转移实验显示,基因沉默组细胞表现了更强的细胞迁移能力;Western blotting结果表明GRHL2的沉默抑制了对Ecadherin和RAB25蛋白的表达,而增加了N-cadherin的表达,GRHL2回补后E-cadherin和RAB25蛋白的表达上升,N-cadherin表达相对下降。结论沉默GRHL2基因后,细胞发生EMT转化,产生失巢凋亡抵抗,导致细胞更易发生侵袭转移;GRHL2基因回补后,细胞发生MET转化,更易发生失巢凋亡,侵袭转移能力下降。Objective To investigate the effect of GRHL2deletion mutant(GRHL2-KO)and complementation(GRHL2-OE)on migration,invasion and anoikis of HCT116cells in human colon cancer.Methods The cell line HCT116,as control,GRHL2-KO and the gene complemented GRHL2-OE which used in this study were obtained from our team in previous research.The mRNA and protein expression of GRHL2were detected by qRT-PCR(Quantitative Real-Time Polymerase Chain Reaction)and western blotting.Anoikis was detected by Calcein AM/EthD-1fluorescence double staining.Cell migration function was observed by wound-healing and transwell assays.Western blot were used to detect the expression of E-cadherin,N-cadherin and RAB25with GRHL2-KO and GRHL2-OE.Results The downregulated mRNA and protein expression of GRHL2in the GRHL2-KO group showed statistically significant difference(P<0.0001)compared to the control group.GRHL2gene complemented group showed notable upregulation of mRNA and protein levels of GRHL2(P<0.0001).Anoikis assay showed that compared with the control group,the survival cell rate increased significantly[Calcein AM:(147.25±20.06)%vs(343.50±6.62)%,P<0.001],while the rate of anoikis cells reduced significantly[EthD-1:(72.12±9.19)%vs(53.96±5.35)%,P<0.01]in the GRHL2-KO group.With GRHL2complementation,the survival rate of cells decreased[Calcein AM:(343.50±6.62)%vs(119.73±14.48)%,P<0.0001],while the rate of anoikis rebounded[EthD-1:(2.25±0.10)%vs(53.96±5.35)%,P<0.001].The cells in the GRHL2-KO group showed stronger cell migration ability in the wound healing and transwell assay.Western blotting results showed that GRHL2silencing inhibited the expression of E-cadherin and RAB25protein,but increased the expression of N-cadherin.After GRHL2complementation,the expression of E-cadherin and RAB25protein increased,and the expression of N-cadherin decreased.Conclusion After silencing the GRHL2gene,the cells underwent EMT transformation,resulting in anoikis resistance,which made the cells more prone to invasion and metastasis.After the GRHL2gene wa

关 键 词：结肠癌 粒状头样2 侵袭转移 失巢凋亡 

分 类 号：R735.3[医药卫生—肿瘤]