Department of Gynecology, Baoji People's Hospital, No. 24 Xinhua Lane, Jing'er Road, Baoji, 721000, Shaanxi Province, China.
Department of Gynecology, Baoji Central Hospital, No. 8 Jiangtan Road, Weibin District, Baoji, 721008, Shaanxi Province, China.
Exp Cell Res. 2024 Sep 1;442(1):114228. doi: 10.1016/j.yexcr.2024.114228. Epub 2024 Aug 26.
Anterior gradient-2 (AGR2) is highly expressed in several tumors and plays an important role in tumor development. However, the biological function of AGR2 in teratomas has not yet been thoroughly studied. In this study, AGR2 was found to be upregulated in teratoma tissues and in human testicular teratoma cell lines by Western blotting and qRT-PCR assays. A DNA Methylation-Specific PCR assay demonstrated that AGR2 upregulation resulted from hypomethylated AGR2 in teratoma cells. NCC-IT and NT2-D1 cells were transfected with pcDNA-AGR2 or sh-AGR2 to obtain AGR2-overexpressed or -silenced cells, and cell proliferation, invasion and glycolysis were determined using CCK-8, 5-ethynyl-2'-deoxyuridine (EdU), Transwell assays, and commercial kits. The results revealed that overexpression of AGR2 promoted teratoma cell proliferation and invasion and elevated glycolysis levels evidencing by the increase in lactate secretion, glucose consumption, ATP levels and the expression of glycolysis-related proteins, while knockdown of AGR2 showed the opposite results. The interactions between AGR2 and annexin A2 (AnXA2), as well as between AnXA2 and epidermal growth factor receptor (EGFR) were verified by co-immunoprecipitation assay. Mechanistic studies revealed that AGR2 interacts with AnXA2 and increases the level of AnXA2 to recruit more AnXA2 to EGFR, there by promoting EGFR expression. A series of rescue experiments showed that knockdown of AnXA2 or EGFR weakened the promotional effects of AGR2 overexpression on the proliferation, invasion, and glycolysis of teratoma cells. Finally, tumorigenicity assays were performed using NT2-D1 cells stably transfected with either LV-NC-shRNA or LV-shAGR2. The results showed that AGR2 knockdown significantly inhibited teratoma tumor growth in vivo. In conclusion, our data suggested that AGR2 facilitates glycolysis in teratomas through promoting EGFR expression by interacting with AnXA2, thereby promoting teratoma cells proliferation and invasion.
AGR2 在多种肿瘤中高表达，在肿瘤发生发展中发挥重要作用。然而，AGR2 在畸胎瘤中的生物学功能尚未得到深入研究。本研究通过 Western blot 和 qRT-PCR 检测发现 AGR2 在畸胎瘤组织和人睾丸畸胎瘤细胞系中上调。DNA 甲基化特异性 PCR 检测表明，畸胎瘤细胞中 AGR2 的上调是由于 AGR2 的低甲基化。使用 pcDNA-AGR2 或 sh-AGR2 转染 NCC-IT 和 NT2-D1 细胞，获得 AGR2 过表达或沉默细胞，通过 CCK-8、5-乙炔基-2'-脱氧尿苷 (EdU)、Transwell 测定和商业试剂盒测定细胞增殖、侵袭和糖酵解。结果表明，AGR2 的过表达促进了畸胎瘤细胞的增殖和侵袭，并提高了糖酵解水平，表现为乳酸分泌、葡萄糖消耗、ATP 水平和糖酵解相关蛋白的表达增加，而 AGR2 的敲低则显示出相反的结果。通过共免疫沉淀实验验证了 AGR2 与膜联蛋白 A2 (AnxA2) 以及 AnxA2 与表皮生长因子受体 (EGFR) 之间的相互作用。机制研究表明，AGR2 与 AnxA2 相互作用，增加 AnxA2 的水平，募集更多的 AnxA2 到 EGFR，从而促进 EGFR 的表达。一系列挽救实验表明，敲低 AnxA2 或 EGFR 削弱了 AGR2 过表达对畸胎瘤细胞增殖、侵袭和糖酵解的促进作用。最后，使用 LV-NC-shRNA 或 LV-shAGR2 稳定转染的 NT2-D1 细胞进行肿瘤生成实验。结果表明，AGR2 敲低显著抑制了体内畸胎瘤肿瘤的生长。综上所述，我们的数据表明，AGR2 通过与 AnxA2 相互作用促进 EGFR 表达，从而促进糖酵解，从而促进畸胎瘤细胞的增殖和侵袭。
