含GATA锌指结构域1对结肠癌细胞迁移的影响

Effect of GATA zinc finger domain containing 1 on migration of colon cancer cells

目的探讨含GATA锌指结构域1(GATAD1)对结肠癌细胞迁移的影响及其机制。方法采用限制性内切酶连接法构建GATAD1干扰质粒SiRNA-GATAD1(Si-GATAD1)。取培养的人正常结肠上皮细胞NCM460及结肠癌细胞Caco-2、HCT-116、HCT-8、HT-29、RKO,应用Western blot法检测细胞中GATAD1蛋白相对表达量,选择其中GATAD1表达水平相对较高的结肠癌细胞HCT-116、RKO用于转染GATAD1干扰质粒Si-GATAD1,另选择其中GATAD1表达水平最低的结肠癌细胞Caco-2用于转染过表达pMZ-GATAD1质粒。取HCT-116、RKO细胞随机分为阴性对照(NC)组和转染Si-GATAD1质粒组(Si-GATAD1组),取Caco-2细胞随机分为NC组、转染过表达pMZ-GATAD1质粒组(pMZ-GATAD1组),Si-GATAD1组HCT-116和RKO细胞分别转染Si-GATAD1质粒,pMZ-GATAD1组Caco-2细转染过表达pMZ-GATAD1质粒,同时将空载体pMZ-MO3质粒转染至NC组HCT-116、RKO和Caco-2细胞。采用细胞划痕实验检测各组细胞划痕愈合率;采用Western blot法检测各组细胞磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路相关蛋白PI3K、Akt、磷酸化蛋白激酶B(p-Akt)、细胞周期蛋白-D1(cyclin D1)的相对表达量。结果结肠癌细胞HCT-8、HCT-116、RKO、HT-29、Caco-2中GATAD1蛋白相对表达量均显著高于人正常结肠上皮细胞NCM460(P<0.05);结肠癌HCT-116、RKO细胞中GATAD1蛋白相对表达量显著高于HCT-8、HCT-29和Caco-2细胞(P<0.05),结肠癌Caco-2细胞中GATAD1蛋白相对表达量显著低于结肠癌HCT-8、HCT-29细胞(P<0.05)。Si-GATAD1组HCT-116、RKO细胞中GATAD1蛋白相对表达量均显著低于NC组(P<0.05),pMZ-GATAD1组Caco-2细胞中GATAD1蛋白相对表达量显著高于NC组(P<0.05)。培养12、24 h,Si-GATAD1组HCT-116、RKO细胞的划痕愈合率均显著低于NC组(P<0.01);pMZ-GATAD1组Caco-2细胞的划痕愈合率均显著高于NC组(P<0.01)。Si-GATAD1组RKO、HCT-116细胞中PI3K、p-Akt、cyclin D1蛋白相对表达量均显著低于其NC组(P<0.05);pMZ-GATAD1组Caco-2细胞中PI3K、p-Akt、cyclin D1蛋白相对表达量均显著高于NC组(P<0.05);Si-GATAD1组RKO、HCT-116细胞及pMZ-GATAD1组Caco-2细胞中Akt蛋白相对表达量与NC组比较差异无统计学意义(P>0.05)。结论GATAD1高表达于结肠癌细胞中,且通过调控PI3K/Akt信号通路促进结肠癌细胞的迁移。

Objective To explore the effect and mechanism of GATA zinc finger domain containing 1(GATAD1)on the migration of colon cancer cells.Methods The GATAD1 interference plasmid SiRNA-GATAD1(Si-GATAD1)was constructed using the restriction endonuclease ligation method.Normal human colon epithelial cells NCM460 and colon cancer cells Caco-2,HCT-116,HCT-8,HT-29,and RKO were cultured separately.The relative expression levels of GATAD1 protein in each group were detected by Western blot.Colon cancer cells HCT-116 and RKO with relatively high GATAD1 expression levels were selected for transfection of GATAD1 interference plasmid Si-GATAD1,and colon cancer cells Caco-2 with the lowest GATAD1 expression levels were selected for transfection of overexpressing pMZ-GATAD1 plasmid.HCT-116 and RKO cells were randomly divided into the negative control group(NC group)and the Si-GATAD1 plasmid transfection group(Si-GATAD1 group).Caco-2 cells were randomly divided into the NC group and the overexpressing pMZ-GATAD1 plasmid transfection group(pMZ-GATAD1 group).Si-GATAD1 plasmids were transfected into HCT-116 and RKO cells,respectively,in the Si-GATAD1 group.The overexpressing pMZ-GATAD1 plasmids were transfected into Caco-2 cells in the pMZ-GATAD1 group.The empty pML-MO3 plasmids were transfected into HCT-116,RKO and Caco-2 cells in the NC group.The cell scratch assay was used to detect the scratch healing rate of cells in each group.Western blot was used to detect the relative expression levels of phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway related proteins PI3K,Akt,phosphorylated protein kinase B(p-Akt),and Cyclin D1(CCND1)in each group.Results The relative expression levels of GATAD1 protein in colon cancer cells HCT-8,HCT-116,RKO,HT-29,and Caco-2 were significantly higher than those in normal human colon epithelial cells NCM460(P<0.05).The relative expression levels of GATAD1 protein in colon cancer cells HCT-116 and RKO were significantly higher than those in HCT-8,HCT-29,and Caco-2 cells(P<0.05),while the relative expression level of GATAD1 protein in Caco-2 cells was significantly lower than that in HCT-8 and HCT-29 cells(P<0.05).The relative expression levels of GATAD1 protein in HCT-116 and RKO cells in the Si-GATAD1 group were significantly lower than those in the NC group(P<0.05),while the relative expression level of GATAD1 protein in Caco-2 cells in the pMZ-GATAD1 group was significantly higher than that in the NC group(P<0.05).After 12 and 24 hours of cultivation,the scratch healing rates of HCT-116 and RKO cells in the Si-GATAD1 group were significantly lower than those in the NC group(P<0.01),while the scratch healing rates of Caco-2 cells in the pMZ-GATAD1 group were significantly higher than those in the NC group(P<0.01).The relative expression levels of PI3K,p-Akt,and CCND1 proteins in RKO and HCT-116 cells in the Si-GATAD1 group were significantly lower than those in the NC group(P<0.05),while the relative expression levels of PI3K,p-Akt,and CCND1 proteins in Caco-2 cells in the pMZ-GATAD1 group were significantly higher than those in the NC group(P<0.05).There was no statistically significant difference in the relative expression of Akt protein between the Si-GATAD1 group(RKO and HCT-116 cells),pMZ-GATAD1 group(Caco-2 cells)and the NC group(P>0.05).Conclusion GATAD1 is highly expressed in colon cancer cells and promotes their migration by regulating the PI3K/Akt signaling pathway.