Supplementary Material for: BIBR1532 affects endometrial cell proliferation, migration and invasion in endometriosis via telomerase inhibition and MAPK signaling

Objectives: The effect of telomerase inhibitor BIBR1532 on endometriotic cells was investigated to explore the inhibitory effect of targeting telomerase on endometriosis. Design: In vitro primary cell culture study. Participants/Materials: Primary endometrial cells derived from eutopic and ectopic endometrium in patients with endometriosis. Setting: University hospital. Methods: Paired eutopic and ectopic endometrial cells were collected from 6 patients from January 2018 to July 2021. A TRAP assay was performed to detect the telomerase activity of the cells. MTT, cell cycle, apoptosis, migration and invasion assays were performed to study the inhibitory effect of BIBR1532. Enrichment analysis was performed to identify the key pathways involved in endometriosis progression and telomerase action. Then, western blotting was used to investigate the expression of related proteins. Results: BIBR1532 treatment significantly inhibited the growth of eutopic and ectopic endometrial cells, with apoptosis and cell cycle signaling involved. Migration and invasion, important characteristics for the establishment of ectopic lesions, were also inhibited by BIBR1532. The MAPK signaling cascade, related to telomerase and endometriosis, was decreased in eutopic and ectopic endometrial stromal cells with the treatment of BIBR1532. Limitations: The severe side effects of telomerase inhibitors might be the main obstacle to clinical application, so it is necessary to find better drug delivery methods in vivo. Conclusions: The telomerase inhibitor BIBR1532 affects endometrial cell proliferation, migration and invasion in endometriosis.

研究目标：本研究探讨端粒酶抑制剂BIBR1532对子宫内膜异位症细胞的作用，以明确靶向端粒酶治疗子宫内膜异位症的抑制效果。研究设计：体外原代细胞培养研究。研究对象与材料：取自子宫内膜异位症患者的在位及异位子宫内膜原代细胞。研究场所：大学附属医院。研究方法：于2018年1月至2021年7月间收集6例患者的配对在位与异位子宫内膜细胞；采用端粒重复序列扩增实验（TRAP）检测细胞端粒酶活性；通过MTT实验、细胞周期检测、细胞凋亡检测、迁移及侵袭实验，探究BIBR1532的细胞抑制作用；开展富集分析以筛选参与子宫内膜异位症进展及端粒酶调控的关键通路；随后采用蛋白质印迹法（Western blotting）检测相关蛋白的表达水平。研究结果：BIBR1532处理可显著抑制在位及异位子宫内膜细胞的增殖，该过程涉及细胞凋亡与细胞周期信号通路；异位病灶建立的关键特征——细胞迁移与侵袭能力，同样受到BIBR1532的抑制。经BIBR1532处理后，与端粒酶及子宫内膜异位症相关的丝裂原活化蛋白激酶（MAPK）信号级联反应，在在位及异位子宫内膜间质细胞中表达下调。研究局限性：端粒酶抑制剂的严重不良反应可能是其临床应用的主要障碍，因此需探索更优的体内给药策略。研究结论：端粒酶抑制剂BIBR1532可影响子宫内膜异位症患者子宫内膜细胞的增殖、迁移及侵袭能力。