Silencing of circ_0007299 suppresses proliferation, migration, and invasiveness and promotes apoptosis of ectopic endometrial stromal cells in endometriosis via miR-424-5p-dependent modulation of CREB1

Background The abnormality of endometrial stromal cells (ESCs) can contribute to endometriosis pathogenesis. Circular RNAs (circRNAs) possess critical roles in endometriosis pathogenesis. Here, we defined the activity and mechanism of human circ_0007299 in the regulation of ectopic ESCs in vitro.

Circ_0007299, miR-424-5p and cAMP response element-binding protein 1 (CREB1) were quantified by qRT-PCR or immunoblotting. Cell viability, proliferation, apoptosis, invasion and motility were gauged by CCK-8, 5-Ethynyl-2ʹ-Deoxyuridine (EdU), flow cytometry, transwell, and wound-healing assays, respectively. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to verify the direct relationship between miR-424-5p and circ_0007299 or CREB1.

Our data showed that circ_0007299 was upregulated in human ectopic endometrium tissues and ectopic ESCs. Silencing endogenous circ_0007299 impeded the proliferation, invasiveness, and motility and enhanced apoptosis of ectopic ESCs. Mechanistically, circ_0007299 regulated miR-424-5p expression. Moreover, circ_0007299 silencing impeded the proliferation, invasiveness, and motility and enhanced apoptosis of ectopic ESCs via its regulation on miR-424-5p. CREB1 was identified as a direct miR-424-5p target, and miR-424-5p overexpression suppressed ectopic ESC proliferation, migration, and invasiveness and promoted apoptosis by downregulating CREB1. Furthermore, circ_0007299 positively modulated CREB1 expression through miR-424-5p competition.

Our findings establish that circ_0007299 silencing impedes the proliferation, invasiveness, and motility and promotes apoptosis of ectopic ESCs at least in part via miR-424-5p-dependent modulation of CREB1. Similar content being viewed by others

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Ethical approval All tissue samples were collected with written informed consent in accordance with the Declaration of Helsinki and with the approval of the Ethics Committee of Gansu University of Traditional Chinese Medicine. Additional information Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary Information Below is the link to the electronic supplementary material. 404_2022_6650_MOESM1_ESM.tif (download TIF ) Supplementary file 1: Supplement Figure 1. Representative immunocytochemical image showing vimentin staining in the isolated ectopic ESCs. n = 3 independent biological replicates. (TIF 354 KB) 404_2022_6650_MOESM2_ESM.tif (download TIF ) Supplementary file 2: Supplement Figure 2. Expression of PCNA and Bax proteins by western blot in ectopic ESCs transfected with si-circ_0007299 or si-NC. n = 3 independent biological replicates. (TIF 879 KB) 404_2022_6650_MOESM3_ESM.tif (download TIF ) Supplementary file 3: Supplement Figure 3. Selection of miR-424-5p and CREB1 in this study. (A) The expression of miR-195-5p, miR-15a-5p and miR-424-5p by qRT-PCR in ectopic ESCs transfected with si-NC or si-circ_0007299. (B) The mRNA expression of KLF12, YAP1, AKT3, TGFBR1, NOTCH2 and CREB1 by qRT-PCR in ectopic ESCs transfected with miR-NC mimic or miR-424-5p mimic. N = 3 independent biological replicates. **P < 0.01, ***P < 0.001, ****P < 0.0001. (TIF 410 KB) 404_2022_6650_MOESM4_ESM.tif (download TIF ) Supplementary file 4: Supplement Figure 4. Effects of CREB1 knockdown on cell proliferation, apoptosis, invasiveness, and motility. (A-G) Ectopic ESCs were transiently transfected with si-CREB1 or si-con. (A) Representative immunoblotting revealing CREB1 protein level in ectopic ESCs transfected as indicated. (B) Cell viability by CCK-8 assay. (C) Cell proliferation by EdU assay. (D) Cell apoptosis by flow cytometry. (E) Transwell assay for cell invasiveness ability. (F) Wound-healing assay for cell migration. (G) Representative immunoblotting showing E-cadherin and N-cadherin expression levels in ectopic ESCs transfected as indicated. n = 6 or 3 independent biological replicates. **P < 0.01, ***P < 0.001, ****P < 0.0001. (TIF 1540 KB) Rights and permissions About this article Cite this article Mao, H., Zhang, X., Yin, L. et al. Silencing of circ_0007299 suppresses proliferation, migration, and invasiveness and promotes apoptosis of ectopic endometrial stromal cells in endometriosis via miR-424-5p-dependent modulation of CREB1. Arch Gynecol Obstet 307, 149–161 (2023). https://doi.org/10.1007/s00404-022-06650-w Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s00404-022-06650-w