Department of Pathology, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 06591, South Korea.
Functional RNomics Research Center, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul, 06591, South Korea.
Gastric Cancer. 2018 Nov;21(6):956-967. doi: 10.1007/s10120-018-0828-8. Epub 2018 Apr 27.
Gastrokine 1 (GKN1) plays important roles in maintaining mucosal homeostasis, and in regulating cell proliferation and differentiation. Here, we determined whether GKN1 is a potential theragnostic marker for gastric cancer.
We identified GKN1 binding proteins using the protein microarray assay and investigated whether GKN1 is one of the exosomal cargo proteins by western blot, immunoprecipitation, and immunofluorescent assays. Cell proliferation and apoptosis were analyzed by MTT, BrdU incorporation, flow cytometry, and western blot assays. We further validated the functional relevance of exosomal GKN1 in MKN1-injected xenograft mice. The possibility of serum GKN1 as a diagnostic marker for gastric cancer was determined by ELISA assay.
In protein microarray assay, GKN1 binding to 27 exosomal proteins was clearly observed. GKN1 was expressed in exosomes derived from HFE-145 gastric epithelial cells by western blot and immunofluorescent assays, but not in exosomes from AGS and MKN1 gastric cancer cells. Exosomes carrying GKN1 inhibited cell proliferation and induced apoptosis in both AGS and MKN1 cells, and exosomes carrying GKN1-treated nude mice-bearing MKN1 xenograft tumors exhibited significantly reduced tumor volume and tumor weight. Silencing of clathrin markedly down-regulated the internalization of exosomal GKN1. Interestingly, serum GKN1 concentrations in patients with gastric cancer were significantly lower than those in healthy individuals and patients with colorectal and hepatocellular carcinomas.
The GKN1 is secreted and internalized in the gastric epithelium by exosome-driven transfer, which inhibits gastric tumorigenesis and supports the clinical application of GKN1 protein in gastric cancer diagnosis and treatment.
胃泌素 1（GKN1）在维持黏膜稳态以及调节细胞增殖和分化方面发挥着重要作用。在这里，我们确定 GKN1 是否是胃癌的一个潜在的治疗标志物。
我们使用蛋白质微阵列分析鉴定了 GKN1 的结合蛋白，并通过 Western blot、免疫沉淀和免疫荧光分析研究了 GKN1 是否是外泌体货物蛋白之一。通过 MTT、BrdU 掺入、流式细胞术和 Western blot 分析来检测细胞增殖和凋亡。我们进一步在 MKN1 注射的异种移植小鼠中验证了外泌体 GKN1 的功能相关性。通过 ELISA 分析确定血清 GKN1 作为胃癌诊断标志物的可能性。
在蛋白质微阵列分析中，清楚地观察到 GKN1 与 27 种外泌体蛋白结合。Western blot 和免疫荧光分析表明 GKN1 在 HFE-145 胃上皮细胞来源的外泌体中表达，但在 AGS 和 MKN1 胃癌细胞来源的外泌体中不表达。携带 GKN1 的外泌体抑制 AGS 和 MKN1 细胞的增殖并诱导其凋亡，并且用携带 GKN1 的外泌体处理的裸鼠携带的 MKN1 异种移植肿瘤的肿瘤体积和重量明显减少。网格蛋白的沉默显著下调了外泌体 GKN1 的内化。有趣的是，胃癌患者的血清 GKN1 浓度明显低于健康个体和结直肠癌、肝细胞癌患者。
GKN1 通过外泌体驱动的转移在胃上皮细胞中被分泌和内化，从而抑制胃肿瘤发生，支持 GKN1 蛋白在胃癌诊断和治疗中的临床应用。
