Department of Pediatric Surgery, Children's Hospital of Nanjing Medical University, Nanjing, China.
Department of Pediatric Surgery, Children's Hospital of Nanjing Medical University, Nanjing, China.
J Pediatr Surg. 2019 Oct;54(10):2032-2037. doi: 10.1016/j.jpedsurg.2019.01.001. Epub 2019 Feb 24.
We previously studied the metabolomics, transcriptomics and proteomics of intestinal tissue of Hirschsprung disease (HSCR) patients; the results suggested that the expression of prostaglandin E2(PGE2), prostaglandin E receptor 2(PTGER2) and microsomal prostaglandin E synthase-1 (mPGES-1) notably increased in HSCR colon tissues. We already verified the differential expression of PGE2/EP2 in HSCR patients; therefore we investigate how mPGES-1 derived PGE2 affects the migration and the potential mechanism in cells, revealing the role of mPGES-1 derived PGE2 in the pathogenesis of Hirschsprung disease.
SH-SY5Y and SK-N-BE2 cell lines were obtained from American Type Culture Collection (ATCC, USA). Prostaglandin E2 and its synthetase inhibitors were purchased from Med Chem Express (MCE, USA). Migration assays were performed with transwell and scratch assays. Cell proliferation was confirmed by CCK8 method. Flow cytometer was used to detect the cell cycle and cell apoptosis. The expressions of mRNA and protein of EP2, ARP2/3 were determined by qRT-PCR and western blot respectively. Immunofluorescence and confocal laser scanning microscopy were used to observe the morphology and function of cytoskeleton.
MPGES-1 derived PGE2 decreased the relative expression of EP2 and ARP2/3 and caused damage to cytoskeleton. As to cell functions, PGE2 inhibited cell migration while having no effects on the proliferation, cell cycle and apoptosis. By adding mPGES-1 inhibitor MK886 the abnormal expression and damaged cell function were reversed.
MPGES-1 derived PGE2 inhibits the cell migration by regulating ARP2/3 complex via prostaglandin E2 receptor. Potential mechanisms are the damage of cytoskeleton and related proteins leading to failure of cell polarize and migration. Here we thoroughly inquire the role mPGES-1 derived PGE2 plays in cell migration which might provide a new thinking in the investigation interrelated to the pathogenesis of HSCR.
我们之前研究了先天性巨结肠症（HSCR）患者的肠道组织的代谢组学、转录组学和蛋白质组学；结果表明，HSCR 结肠组织中前列腺素 E2（PGE2）、前列腺素 E 受体 2（PTGER2）和微粒体前列腺素 E 合酶-1（mPGES-1）的表达显著增加。我们已经验证了 PGE2/EP2 在 HSCR 患者中的差异表达；因此，我们研究了 mPGES-1 衍生的 PGE2 如何影响细胞的迁移及其潜在机制，揭示了 mPGES-1 衍生的 PGE2 在 HSCR 发病机制中的作用。
SH-SY5Y 和 SK-N-BE2 细胞系购自美国典型培养物保藏中心（ATCC，美国）。前列腺素 E2 和其合成酶抑制剂购自 Med Chem Express（MCE，美国）。使用 Transwell 和划痕实验进行迁移实验。通过 CCK8 法证实细胞增殖。流式细胞仪用于检测细胞周期和细胞凋亡。通过 qRT-PCR 和 Western blot 分别测定 EP2、ARP2/3 的 mRNA 和蛋白表达。免疫荧光和共聚焦激光扫描显微镜用于观察细胞骨架的形态和功能。
mPGES-1 衍生的 PGE2 降低了 EP2 和 ARP2/3 的相对表达，并导致细胞骨架受损。就细胞功能而言，PGE2 抑制细胞迁移，而对增殖、细胞周期和凋亡没有影响。通过添加 mPGES-1 抑制剂 MK886，异常表达和受损的细胞功能得到逆转。
mPGES-1 衍生的 PGE2 通过调节前列腺素 E2 受体的 ARP2/3 复合物抑制细胞迁移。潜在机制是细胞骨架和相关蛋白的损伤导致细胞极化和迁移失败。在这里，我们深入探究了 mPGES-1 衍生的 PGE2 在细胞迁移中的作用，这可能为研究与 HSCR 发病机制相关的问题提供新的思路。
