PDF(Pubmed)
Abstract：
BACKGROUND: Wnt/FZD-mediated signaling pathways are activated in more than 90% of hepatocellular carcinoma (HCC) cell lines. As a well-known secretory glycoprotein, Wnt3 can interact with FZD receptors on the cell surface, thereby activating the Wnt/β-catenin signaling pathway. However, the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear.
OBJECTIVE: To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.
METHODS: Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites, actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation, the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy, and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.
RESULTS: Wnt3 has two N-glycosylation-modified sites (Asn90 and Asn301); when a single site at amino acid 301 is mutated, the stability of Wnt3 is weakened; the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously; and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated. Cell proliferation, migration and invasion are also weakened in the case of single 301 site and double-site mutations.
CONCLUSIONS: These results indicate that by inhibiting the N-glycosylation of Wnt3, the proliferation, migration, invasion and colony formation abilities of liver cancer cells can be weakened, which might provide new therapeutic strategies for clinical liver cancer in the future.
OBJECTIVE: To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells.
METHODS: Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites, actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation, the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy, and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments.
RESULTS: Wnt3 has two N-glycosylation-modified sites (Asn90 and Asn301); when a single site at amino acid 301 is mutated, the stability of Wnt3 is weakened; the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously; and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated. Cell proliferation, migration and invasion are also weakened in the case of single 301 site and double-site mutations.
CONCLUSIONS: These results indicate that by inhibiting the N-glycosylation of Wnt3, the proliferation, migration, invasion and colony formation abilities of liver cancer cells can be weakened, which might provide new therapeutic strategies for clinical liver cancer in the future.
摘要：
背景：Wnt/FZD介导的信号通路在超过90％的肝细胞癌（HCC）细胞系中被激活。作为一种众所周知的分泌糖蛋白，Wnt3可以与细胞表面的FZD受体相互作用，从而激活Wnt/β-catenin信号通路。然而,Wnt3的N-糖基化修饰位点及其对蛋白质生物学功能的影响尚不清楚。
目的：探讨Wnt3N-糖基化对肝癌细胞生物学功能的影响。
方法：使用定点诱变来验证Wnt3N-糖基化位点，放线菌素D处理用于检测定点突变后Wnt3的稳定性，通过激光共聚焦显微镜观察到N-糖基化位点定向突变体Wnt3与FZD7的结合，并通过westernblot和细胞功能实验检测Wnt3的N-糖基化位点定向突变对Wnt/β-catenin信号通路和HCC细胞进展的影响。
结果：Wnt3具有两个N-糖基化修饰的位点（Asn90和Asn301）；当氨基酸301处的单个位点发生突变时，Wnt3的稳定性减弱；当两个位点同时突变时，Wnt3与FZD7的结合能力降低；与Wnt/β-catenin信号通路相关的蛋白水平下调。细胞增殖，在单301位点和双位点突变的情况下，迁移和侵袭也减弱。
结论：这些结果表明，通过抑制Wnt3的N-糖基化，迁移,肝癌细胞的侵袭和集落形成能力可以减弱，这可能为未来临床肝癌提供新的治疗策略。
目的：探讨Wnt3N-糖基化对肝癌细胞生物学功能的影响。
方法：使用定点诱变来验证Wnt3N-糖基化位点，放线菌素D处理用于检测定点突变后Wnt3的稳定性，通过激光共聚焦显微镜观察到N-糖基化位点定向突变体Wnt3与FZD7的结合，并通过westernblot和细胞功能实验检测Wnt3的N-糖基化位点定向突变对Wnt/β-catenin信号通路和HCC细胞进展的影响。
结果：Wnt3具有两个N-糖基化修饰的位点（Asn90和Asn301）；当氨基酸301处的单个位点发生突变时，Wnt3的稳定性减弱；当两个位点同时突变时，Wnt3与FZD7的结合能力降低；与Wnt/β-catenin信号通路相关的蛋白水平下调。细胞增殖，在单301位点和双位点突变的情况下，迁移和侵袭也减弱。
结论：这些结果表明，通过抑制Wnt3的N-糖基化，迁移,肝癌细胞的侵袭和集落形成能力可以减弱，这可能为未来临床肝癌提供新的治疗策略。
