摘要
目的研究G补缀FHA域血管新生因子1(angiogenic factor with G-patch and FHA domain 1,AGGF1)调控Wnt/β-catenin信号通路对结直肠癌细胞(colorectal cancer,CRC)增殖、侵袭和上皮间质转化(epithelial mesenchymal transition,EMT)的可能机制。方法通过裸鼠荷瘤免疫组化法检测AGGF1在正常组织和癌组织中的表达。qRT-PCR和Western blotting检测NCM460、SW620、HT29、HCT116、SW480中AGGF1的mRNA和蛋白表达水平。于HCT116细胞中过表达AGGF1,分为Vector组和AGGF1组;于SW620细胞中敲减AGGF1,分为shControl组和shAGGF1组。CCK-8法和克隆形成试验测定细胞活性和细胞克隆数目。划痕试验和Transwell试验检测细胞迁移和侵袭情况。免疫荧光检测细胞E-cadherin、N-cadherin的表达。Western blotting检测细胞E-cadherin、N-cadherin、AGGF1、β-catenin、糖原合酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)、p-GSK-3β蛋白表达水平。结果AGGF1蛋白表达在CRC组织中明显增高。AGGF1 mRNA和蛋白表达水平在HCT116细胞中最低,在SW620细胞中最高。CCK-8法、克隆形成试验、划痕试验和Transwell试验结果显示,在HCT116细胞中,与Vector组比较,AGGF1组48,72,96 h细胞活性、克隆细胞数目、相对迁移距离、侵袭细胞数显著增加(P<0.05或P<0.01);而在SW620细胞中,与shControl组比较,shAGGF1组结果相反(P<0.05或P<0.01)。免疫荧光和Western blotting检测结果显示,与Vector组比较,AGGF1组E-cadherin荧光表达和蛋白表达水平降低(P<0.01),N-cadherin增强(P<0.05或P<0.01),同时上调AGGF1、β-catenin、p-GSK-3β/GSK-3β蛋白表达(P<0.01);与shControl组比较,shAGGF1组E-cadherin荧光表达和蛋白表达水平显著升高(P<0.01),N-cadherin显著降低(P<0.05或P<0.01),同时下调AGGF1、β-catenin、p-GSK-3β/GSK-3β蛋白表达(P<0.05或P<0.01)。结论AGGF1可通过激活Wnt/β-catenin信号通路异常表达,上调β-catenin、p-GSK-3β、N-cadherin表达,下调E-cadherin表达,促进CRC细胞增殖、迁移、侵袭和EMT
OBJECTIVE To investigate the possible mechanism of angiogenic factor with G-patch and FHA domain(AGGF1)regulating Wnt/β-catenin signaling pathway on proliferation,invasion and epithelial mesenchymal transition(EMT)of colorectal cancer(CRC).METHODS The expression of AGGF1 in normal tissues and tumor tissues was detected by immunohistochemistry of tumor bearing nude mice.The expression of AGGF1 mRNA and protein in NCM460,SW620,HT29,HCT116 and SW480 were detected by qRT-PCR and Western blotting.AGGF1 was overexpressed in HCT116 cells and divided into Vector group and AGGF1 group,AGGF1 was knocked down in SW620 cells and divided into shControl group and shAGGF1 group by cell transfection.CCK-8 assay and colony formation assay were used to determine the cell viability and the number of cell clones.Cell migration and invasion were detected by wound healing assay and Transwell test.The expression of E-cadherin and N-cadherin were detected by immunofluorescence.Western blotting was used to detect the expression of E-cadherin,N-cadherin,AGGF1,β-catenin,glycogen synthase kinase-3β(GSK-3β)and p-GSK-3β.RESULTS The expression of AGGF1 protein was significantly increased in CRC.The expression levels of AGGF1 mRNA and protein in HCT116 cells were the lowest and the highest in SW620 cells.CCK-8 assay,clone formation assay,wound healing assay and Transwell test showed that in HCT116 cells,compared with the Vector group,the activity of cells at 48,72 and 96 h,the number of cloned cells,the relative migration distance and the number of invasive cells in AGGF1 group were significantly increased(P<0.05 or P<0.01);in SW620 cells,compared with the shControl group,the shAGGF1 group had the opposite results(P<0.05 or P<0.01).The results of immunofluorescence and Western blotting showed that compared with the Vector group,E-cadherin fluorescence and protein expression were decreased in AGGF1 group(P<0.01),N-cadherin was increased(P<0.05 or P<0.01),meanwhile,AGGF1,β-catenin,p-GSK-3β/GSK-3βprotein expression was up-regulated(P<0.01)
作者
杨鹏
吴捷
陈文斌
YANG Peng;WU Jie;CHEN Wenbin(The First Affiliated Hospital,Zhejiang University,School of Medicine,Hangzhou 310003,China;The Fifth People’s Hospital of Yuhang District,Hangzhou 311100,China)
出处
《中国现代应用药学》
CAS
CSCD
北大核心
2021年第11期1319-1326,共8页
Chinese Journal of Modern Applied Pharmacy
