Center for Reproductive Medicine, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, China.
Biomed Res Int. 2022 Oct 25;2022:6006981. doi: 10.1155/2022/6006981. eCollection 2022.
Fyn has been proven to be involved in various cell behaviors and pathophysiological processes. However, the expression and roles of Fyn in trophoblasts remain unclear. Here, we aimed to evaluate the participation of Fyn in trophoblast behavior and function, and the related mechanisms were briefly explored. Fyn expression in the HTR-8/SVneo, JEG-3, and JAR cell lines was evaluated by immunofluorescence, quantitative real-time PCR and western blotting. Fyn expression in human hydatidiform moles was also determined by immunohistochemistry and western blot. To explore the effects of Fyn, HTR-8/SVneo and JEG-3 cells were transfected with Fyn shRNA or overexpression plasmid or treated with the Fyn activity inhibitor SU6656 or ERK1/2 inhibitor U0126. The migration, proliferation, and apoptosis of trophoblast cells were assessed using transwell assays, flow cytometry, and cell counting kit-8 assays, respectively. The production of primary inflammatory cytokines, HLA-G and active matrix metallopeptidase (MMP) 2/9, and the phosphorylation of ERK1/2 and STAT3 were evaluated by ELISA, western blot, or gelatin zymography. The results showed that Fyn was expressed by trophoblast cells, mainly in the cytoplasm and membrane. Fyn expression and activity levels both increased in order from HTR-8/SVneo and JAR to JEG-3. The overexpression of Fyn promoted the proliferation and migration of trophoblast cells and inhibited their apoptosis, while the opposite effects were observed for Fyn knockdown and inhibition. Fyn regulated inflammatory cytokine production in trophoblast cells by promoting TGF- and IL-4 secretion while inhibiting IFN- and TNF- secretion. Moreover, HLA-G expression in JEG-3 was positively regulated by Fyn. Fyn also facilitated the expression of active MMP2/9 and the activation of ERK1/2 and STAT3. Besides, it was confirmed that Fyn regulated trophoblast cell activities through ERK1/2 signal pathway by using U0126. Our study first detected the expression of Fyn in trophoblast cells. Fyn played pivotal roles in trophoblast cell behaviors and function, ERK1/2 was one of its targets, and MMP2/9 and STAT3 may also be involved in the regulatory mechanism.
Fyn 已被证实参与多种细胞行为和生理病理过程。然而，Fyn 在滋养细胞中的表达和作用尚不清楚。本研究旨在评估 Fyn 在滋养细胞行为和功能中的作用，并初步探讨相关机制。通过免疫荧光、实时定量 PCR 和 Western blot 检测 HTR-8/SVneo、JEG-3 和 JAR 细胞系中 Fyn 的表达。通过免疫组化和 Western blot 检测人葡萄胎组织中 Fyn 的表达。为了研究 Fyn 的作用，用 Fyn shRNA 或过表达质粒转染 HTR-8/SVneo 和 JEG-3 细胞，或用 Fyn 活性抑制剂 SU6656 或 ERK1/2 抑制剂 U0126 处理细胞。用 Transwell 实验、流式细胞术和细胞计数试剂盒分别评估滋养细胞的迁移、增殖和凋亡。用 ELISA、Western blot 或明胶酶谱法评估原代炎症细胞因子、HLA-G 和活性基质金属蛋白酶（MMP）2/9 的产生以及 ERK1/2 和 STAT3 的磷酸化。结果表明，Fyn 由滋养细胞表达，主要位于细胞质和细胞膜。Fyn 的表达和活性水平均从 HTR-8/SVneo 和 JAR 逐渐增加到 JEG-3。Fyn 过表达促进滋养细胞的增殖和迁移，抑制凋亡，而 Fyn 敲低和抑制则产生相反的效果。Fyn 通过促进 TGF-β和 IL-4 的分泌，抑制 IFN-和 TNF-α的分泌，调节滋养细胞中炎症细胞因子的产生。此外，Fyn 正向调节 JEG-3 中 HLA-G 的表达。Fyn 还促进活性 MMP2/9 的表达和 ERK1/2 和 STAT3 的激活。此外，通过使用 U0126 证实 Fyn 通过 ERK1/2 信号通路调节滋养细胞的活性。本研究首次检测了 Fyn 在滋养细胞中的表达。Fyn 在滋养细胞行为和功能中发挥关键作用，ERK1/2 是其靶点之一，MMP2/9 和 STAT3 可能也参与了调控机制。
