OBJECTIVE To investigate the role(s) of hyaluronan synthase 2 (HAS2) and hyaluronan in disease progression of endometriosis and epidermal growth factor (EGF)-induced motility changes of endometriotic cells. DESIGN A case-control experimental study and in vitro primary cell culture study. SETTING University hospital-affiliated research centers. PATIENTS A total of 21 women with stage I/II endometriosis, 33 women with stage III/IV endometriosis with endometrioma, and 32 women without endometriosis were included in our study. INTERVENTIONS Serum, eutopic endometrial tissues, and/or ectopic endometriotic tissues were collected. Primary eutopic endometrial stromal cells (EuESCs) and ectopic ovarian endometriotic stromal cells (OvESCs) were isolated and cultured from women with ovarian endometrioma, and then treated with or without EGF. MAIN OUTCOME MEASURES The concentrations of EGF and hyaluronan in serum were analyzed by enzyme-linked immunosorbent assay. The expressions and localizations of EGF receptor (EGFR), phosphorylated-(p)EGFR, HAS2, and hyaluronan receptor CD44 in tissues were examined by immunohistochemistry. The mRNA and protein levels of HAS2 in EuESCs and OvESCs were examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot, respectively, and the concentrations of hyaluronan in conditioned medium were examined by enzyme-linked immunosorbent assay (ELISA). Cell motility was evaluated by transwell migration/invasion assays. RESULTS Serum EGF and hyaluronan concentrations were higher in women with stage III/IV endometriosis than in women with stage I/II or without endometriosis. EGFR, pEGFR, HAS2, and CD44 were immunolocalized in eutopic endometrium and ectopic endometriotic lesions, and the expressions of pEGFR and HAS2 were elevated in ectopic endometriotic lesions compared to eutopic endometrium. Treatment with EGF upregulated HAS2 and hyaluronan expression as well as cell migration and invasion in both EuESCs and OvESCs, and pharmaceutical blocking of EGFR abolished these effects. In addition, knockdown of HAS2 by small interfering RNA attenuated both basal and EGF-induced hyaluronan expression and cell motility changes. Notably, ERK1/2 and AKT signaling pathways were shown to be downstream of EGF in regulating HAS2 and hyaluronan expression as well as cell migration and invasion. CONCLUSION EGF increased the expression of endometriosis-associated hyaluronan and its synthase HAS2, both of which mediated EGF-induced stromal cell migration and invasion in women with endometriosis.

中文翻译：

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表皮生长因子通过上调透明质酸合成酶 2 和透明质酸促进子宫内膜异位症中的基质细胞迁移和侵袭

目的探讨透明质酸合酶2（HAS2）和透明质酸在子宫内膜异位症的疾病进展和表皮生长因子（EGF）诱导的子宫内膜异位症细胞运动性变化中的作用。设计 病例对照实验研究和体外原代细胞培养研究。设置大学附属研究中心。患者 共有 21 名患有 I/II 期子宫内膜异位症的女性、33 名患有 III/IV 期子宫内膜异位症的女性和 32 名没有子宫内膜异位症的女性被纳入我们的研究。干预措施 收集血清、在位子宫内膜组织和/或异位子宫内膜组织。从患有卵巢子宫内膜异位症的女性中分离和培养原代在位子宫内膜基质细胞 (EuESCs) 和异位卵巢子宫内膜异位基质细胞 (OvESCs)，然后使用或不使用 EGF 进行治疗。主要观察指标采用酶联免疫吸附法分析血清中EGF和透明质酸的浓度。通过免疫组织化学检查组织中 EGF 受体 (EGFR)、磷酸化-(p)EGFR、HAS2 和透明质酸受体 CD44 的表达和定位。分别通过逆转录-定量聚合酶链反应 (RT-qPCR) 和蛋白质印迹法检测 EuESCs 和 OvESCs 中 HAS2 的 mRNA 和蛋白质水平，并通过酶联免疫吸附试验 (ELISA) 检测条件培养基中透明质酸的浓度）。通过跨孔迁移/侵袭测定评估细胞运动性。结果 III/IV 期子宫内膜异位症女性的血清 EGF 和透明质酸浓度高于 I/II 期或无子宫内膜异位症女性。EGFR、pEGFR、HAS2、CD44和CD44免疫定位于在位子宫内膜和异位子宫内膜病变中，与在位子宫内膜相比，异位子宫内膜病变中pEGFR和HAS2的表达升高。EGF 治疗上调了 HAS2 和透明质酸的表达以及 EuESC 和 OvESC 中的细胞迁移和侵袭，并且药物阻断 EGFR 消除了这些影响。此外，小干扰 RNA 对 HAS2 的敲低减弱了基础和 EGF 诱导的透明质酸表达和细胞运动性变化。值得注意的是，ERK1/2 和 AKT 信号通路被证明在 EGF 的下游调节 HAS2 和透明质酸的表达以及细胞迁移和侵袭。结论 EGF 增加了子宫内膜异位症相关透明质酸及其合酶 HAS2 的表达，