Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250012, PR China.
Department of Obstetrics and Gynecology, Qilu Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250012, PR China.
Reprod Biomed Online. 2020 Nov;41(5):790-800. doi: 10.1016/j.rbmo.2020.04.022. Epub 2020 May 15.
Does the aggregation of M2 macrophages affect the expression of gene associated with retinoid-interferon-induced mortality 19 (GRIM-19) in adenomyosis?
Endometrial tissues were collected from patients with (n = 15) and without (n = 15) adenomyosis. Tissues were analysed for GRIM-19 and toll-like receptor 4 (TLR4) expression by immunohistochemistry and western blotting. Apoptosis was analysed by TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. Human endometrial stromal cells (HESC) were transfected with GRIM-19 small interfering RNA (SiRNA) to knockdown GRIM-19 expression. The HESC were co-cultured with M2 macrophages to detect the influence of M2 macrophages in HESC cells. Analyses included GRIM-19, caspase-3 and TLR4 expression by western blotting, and GRIM-19 and TLR4 by quantitative real-time polymerase chain reaction. Apoptosis was measured by flow cytometry and TUNEL assay. Cell proliferation (Cell Counting Kit-8 assay) and migration assays were carried out.
The expression of GRIM-19 was significantly lower in adenomyosis lesions compared with controls (P < 0.001). Deficiency of GRIM-19 induced by siRNA decreased apoptosis and increased proliferation and migration in HESC. A significant decrease in GRIM-19 expression occurred in HESC after co-culture with M2 macrophages (P = 0.018). After co-culture with M2 macrophage, apoptosis decreased and proliferation and cell invasion in HESC increased. Protein (P = 0.006) and mRNA (P = 0.013) expression of TLR4 in HESC also reduced after this co-culture. Up-regulation of GRIM-19 occurred in HESC treated with the activator TLR4 (P = 0.016). Up-regulation of GRIM-19 was significantly reversed in cells treated with the TLR4 inhibitor (P = 0.011).
M2 macrophages may be involved in regulating the expression of GRIM-19 partly through the TLR4 signalling axis in adenomyosis.
M2 巨噬细胞的聚集是否会影响腺肌病中与维甲酸-干扰素诱导的死亡率 19（GRIM-19）相关基因的表达？
从患有（n=15）和不患有（n=15）腺肌病的患者中收集子宫内膜组织。通过免疫组织化学和蛋白质印迹分析组织中 GRIM-19 和 toll 样受体 4（TLR4）的表达。通过末端脱氧核苷酸转移酶介导的 dUDP 缺口末端标记（TUNEL）分析检测细胞凋亡。用人子宫内膜基质细胞（HESC）转染 GRIM-19 小干扰 RNA（siRNA）以敲低 GRIM-19 表达。将 HESC 与 M2 巨噬细胞共培养，以检测 M2 巨噬细胞对 HESC 细胞的影响。分析包括通过蛋白质印迹检测 GRIM-19、半胱天冬酶-3 和 TLR4 的表达，通过实时定量聚合酶链反应检测 GRIM-19 和 TLR4。通过流式细胞术和 TUNEL 分析测量细胞凋亡。进行细胞增殖（细胞计数试剂盒-8 测定）和迁移测定。
与对照组相比，腺肌病病变中 GRIM-19 的表达明显降低（P<0.001）。siRNA 诱导的 GRIM-19 缺陷降低了 HESC 的细胞凋亡，并增加了其增殖和迁移。与 M2 巨噬细胞共培养后，HESC 中 GRIM-19 的表达明显降低（P=0.018）。与 M2 巨噬细胞共培养后，HESC 的细胞凋亡减少，增殖和细胞侵袭增加。与 M2 巨噬细胞共培养后，HESC 中 TLR4 的蛋白质（P=0.006）和 mRNA（P=0.013）表达也降低。用 TLR4 激动剂处理 HESC 后，GRIM-19 的表达上调（P=0.016）。用 TLR4 抑制剂处理细胞后，GRIM-19 的上调显著逆转（P=0.011）。
M2 巨噬细胞可能通过腺肌病中 TLR4 信号轴参与调节 GRIM-19 的表达。
