AGGF1通过激活Wnt/β-catenin信号通路促进结直肠癌发生发展

AGGF1 Promotes Colorectal Cancer Progression by Activating Wnt/β-catenin Signaling Pathway

关键词:  AGGF1  Wnt/β-catenin信号通路  结直肠癌  细胞增殖  侵袭  上皮间质转化

Key words:  AGGF1  wnt/β-catenin signaling pathway  colorectal cancer  cell proliferation  invasion  epithelial mesenchymal transition

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，从而促进CRC发生发展。

Abstract:

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-3β protein expression was up-regulated(P<0.01). Compared with shControl group, E-cadherin fluorescence and protein expression were increased in shAGGF1 group(P<0.01), N-cadherin was decreased (P<0.05 or P<0.01), and downregulation of AGGF1, β-catenin, p-GSK-3β/GSK-3β protein expression(P<0.05 or P<0.01). CONCLUSION AGGF1 can stimulate the abnormal expression of Wnt/β-catenin signaling pathway, up regulate the expression of β-catenin, p-GSK-3β, N-cadherin, down regulate the expression of E-cadherin, to promote the proliferation, migration, invasion and EMT of CRC cells, thus accerelating the occurrence and development of CRC.