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

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 was complemented,the cells showed the properties of MET transformation,more prone to anoikis,and decreased ability of invasion and metastasis.