APEX1介导Jagged1上调驱动结肠癌进展的机制

APEX1 Mediates the Mechanism of Jagged Up- regulation ThatDrives the Progression of Colon Cancer

目的探讨APEX1介导Jagged1上调驱动结肠癌进展的机制。方法APEX1 siRNA转染NCI-H548和DLD1,构建APEX1低表达细胞;SW480和HT29转染APEX1 shRNA,构建APEX1超表达细胞。NCI-H548细胞用于构建APEX1超表达、Jagged1低表达、Jagged1超表达。将构建的NCI-H548-APEX1、NCI-H548-APEX1+Jagged1siRNA,NCI-H548-APEX1+Jagged1细胞注射小鼠体内用于肿瘤生长检测。通过Western blot分析人结肠癌细胞系中APEX1、Jagged1、Notch1和Notch3的蛋白表达。通过MTT试验检测细胞增殖。通过Transwell小室试验检测细胞迁移侵袭。用指定细胞的上清液培养HUVECs成管进行血管生成试验。通过卡尺测量异种移植小鼠肿瘤大小。结果NCI-H548和DLD1中APEX1、Jagged1、Notch1和Notch3蛋白表达较SW480、HT29升高(P<0.05),在APEX1高表达的NCI-H548和DLD1中,Jagged1和Notch蛋白表达较高。相反,在APEX1低表达的SW480、HT29中,Jagged1和Notch蛋白表达较低。在NCI-H548、DLD1等结肠癌细胞中转染APEX1 siRNA敲低APEX1后,Jagged1蛋白和/Notch蛋白水平显著降低(P<0.05),而在SW480-APEX1和HT29-APEX1细胞中,Jagged1蛋白/Notch蛋白平显著升高(P<0.05)。NCI-H548-APEX1+Jagged1 siRNA细胞增殖较NCI-H548-APEX1降低(P<0.05),NCI-H548-APEX1+Jagged1细胞增殖较NCI-H548-APEX1+Jagged1 siRNA升高(P<0.05)。NCI-H548-APEX1+Jagged1 siRNA细胞迁移、侵袭能力较NCI-H548-APEX1降低(P<0.05),NCI-H548-APEX1+Jagged1细胞迁移侵袭能力较NCI-H548-APEX1+Jagged1siRNA升高(P<0.05)。NCI-H548-APEX1+Jagged1 siRNA细胞血管生成量较NCI-H548-APEX1减少(P<0.05),NCI-H548-APEX1+Jagged1细胞血管生成量较NCI-H548-APEX1+Jagged1 siRNA增多(P<0.05)。注射NCI-H548-APEX1+Jagged1 siRNA细胞的小鼠肿瘤体积较注射NCI-H548-APEX1细胞减小(P<0.05),注射NCI-H548-APEX1+Jagged1细胞的小鼠肿瘤体积较注射NCI-H548-APEX1+Jagged1 siRNA细胞增大(P<0.05)。结论APEX1通过上调Jagged1的表达,正向调节Notch信号通路,从而促进结肠癌在体内和体外的肿瘤发生。由于APEX1、Jagged1和cleaved Notch的水平在人类结肠癌细胞中密切相关,因此通过强烈激活Jagged1/Notch信号,药物靶向APEX1过表达可能是治疗或预防结肠癌的一种很有前景的方法。

Objective To explore the mechanism of APEX1-mediated Jagged up-regulation driving the progression of colon cancer.Methods APEX1 siRNA was transfected with NCI-H548 and DLD1 to construct APEX1 cells with low expression.SW480 and HT29 were transfected with APEX1 shRNA to construct APEX1 overexpressing cells.NCI-H548 cells were used to construct APEX1 overexpression,Jagged1 under-expression and Jagged1 overexpression.The constructed NCI-H548-APEX,NCI-H548-APEX1+Jagged1 siRNA and NCI-H548-APEX1+Jagged1 cells were injected into mice for tumor growth detection.Protein expressions of APEX1,Jagged1,Notch1 and Notch3 in human colon cancer cell lines were measured by western blot analysis.Cell proliferation was detected by MTT assay.Cell migration and invasion were detected by Trans-well assay.HUVECs were cultured in tubes with the supernatant of designated cells for angiogenesis tests.The tumor size of xenografted mice was measured by calipers.Results The expressions of APEX1,Jagged1,Notch1 and Notch3 in NCI-H548 and DLD1 were higher than those in SW480 and HT29(P<0.05).The expressions of Jagged1 and Notch were higher in NCI-H548 and DLD1 with high APEX1 expression.On the contrary,Jagged1 and Notch protein expressions were lower in SW480 and HT29 with low APEX1 expression.After APEX1 siRNA knockdown of APEX1 in colon cancer cells,such as NCI-H548 and DLD1,Jagged1 protein and Notch protein levels were significantly decreased(P<0.05).However,in sw40-APEX1 and HT29-APEX1 cells,Jagged1 protein and Notch protein were significantly increased(P<0.05).The proliferation of siRNA cells of NCI-H548-APEX1+Jagged1 was lower than that of NCI-H548-APEX1(P<0.05).Cell proliferation of NCI-H548-APEX1+Jagged1 was higher than that of NCI-H548-APEX1+Jagged1 siRNA(P<0.05).NCI-H548-APEX1+Jagged1 siRNA cell migration and invasion ability decreased compared with NCI-H548-APEX1(P<0.05).The migration and invasion ability of NCI-H548-APEX1+Jagged1 cells was higher than that of NCI-H548-APEX1+Jagged1 siRNA(P<0.05).The angiogenesis of NCI-H548-APEX1+Jagged1 siRNA cells was lower than that of NCI-H548-APEX1(P<0.05).The siRNA volume of NCI-H548-APEX1+Jagged1 cells was higher than that of NCI-H548-APEX1+Jagged1 cells(P<0.05).The tumor volume of mice injected with NCI-H548-APEX1+Jagged1 siRNA cells was decreased compared with that injected with NCI-H548-APEX1 cells(P<0.05).The tumor volume of mice injected with NCI-H548-APEX1+Jagged1 cells was increased compared with those injected with NCI-H548-APEX1+Jagged1 siRNA cells(P<0.05).Conclusion APEX1 can positively regulate the Notch signaling pathway by up-regulating the expression of Jagged1,thus promoting the tumorigenesis of colon cancer both in vivo and in vitro.Because APEX1,Jagged1,and cleaved Notch levels are closely correlated in human colon cancer cells,drug targeting APEX1 overexpression by strongly activating Jagged1/Notch signaling could serve as a promising approach for the treatment or prevention of colon cancer.