Long noncoding RNA ADAMTS9-AS1 represses ferroptosis of endometrial stromal cells by regulating the miR-6516-5p/GPX4 axis in endometriosis

www.nature.com/scientificreports OPEN Long noncoding RNA ADAMTS9‑AS1 represses ferroptosis of endometrial stromal cells by regulating the miR‑6516‑5p/GPX4 axis in endometriosis Yiting Wan1,2, Cancan Gu1,2, Jueying Kong1, Jin Sui1, Ling Zuo1, Yanhua Song1* & Jing Chen1* Endometriosis (EMs) is one of the most frequent diseases of reproductive-age women and is characterized by the growth of endometrial tissues beyond the uterus. The enhanced proliferative and migratory potential of endometrial stromal cells (ESCs) plays an important role in the progression of EMs. Mounting studies have demonstrated that long noncoding RNAs (lncRNAs) exert an important role in regulating the development and progression of EMs. Given the aberrant expression of lncRNA ADAMTS9-AS1 in ectopic endometrium (ecEM), we investigated the biological effect of ADAMTS9-AS1 on ESC proliferation and migration and explored the underlying mechanism. The current data showed that ADAMTS9-AS1 expression was significantly upregulated in ecEM compared with eutopic endometrium (euEM) in patients with EMs and in a murine model of EMs. Functionally, ADAMTS9-AS1 knockdown in ectopic ESCs (EESCs) decreased cell viability and migration, whereas ADAMTS9-AS1 overexpression in normal ESCs (NESCs) enhanced cell viability and migration. More importantly, the effect of ADAMTS9-AS1 inhibition on decreasing ESC viability was significantly blocked by ferrostatin-1 (Fer-1, a ferroptosis inhibitor), and ADAMTS9-AS1 overexpression repressed erastin (a ferroptosis activator)-induced cell death. Furthermore, the regulatory role of ADAMTS9-AS1 in ferroptosis was defined and evidenced by increased reactive oxygen species (ROS) levels and malonyl dialdehyde (MDA) content and decreased expression of glutathione peroxidase 4 (GPX4) after ADAMTS9-AS1 inhibition. Mechanistically, ADAMTS9-AS1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-6516-5p to derepress the expression of GPX4, the critical repressor of ferroptosis. Taken together, these results demonstrate that upregulated ADAMTS9-AS1 accelerates ESC proliferation and migration by regulating miR-6516-5p/GPX4-dependent ferroptosis and may be a potential target for the treatment of EMs. Endometriosis (EMs) is a common and polyfactorial disorder in which environmental, genetic, immunologic, and hormonal aberrations are correlated with the pathological process of E Ms1,2. EMs is characterized by the growth of histologically normal endometrial tissues beyond the uterus and affects between 10 and 15% of women of fertile age w orldwide3,4. The major clinical manifestations of EMs include infertility, chronic pelvic pain and vaginal bleeding and thus negatively affect the quality of life of these women. Recently, mounting experimental evidence has demonstrated that noncoding RNAs (ncRNAs) play an important regulatory role in the pathogenesis of EMs and in EMs-associated s ymptoms5,6. Long noncoding RNA (lncRNA) is a kind of RNA molecule with a length of more than 200 nt that does not possess protein-coding potential7. An increasing number of studies have suggested that dysregulated lncRNA 1 Department of Gynecology, Shanghai Municipal Hospital of Traditional Chinese Medicine, Shanghai University of Traditional Chinese Medicine, No. 274 Middle Zhijiang Road, Shanghai 200071, China. 2These authors contributed equally: Yiting Wan and Cancan Gu. *email: ; Scientific Reports | (2022) 12:2618 | https://doi.org/10.1038/s41598-022-04963-z 1 Vol.:(0123456789) www.nature.com/scientificreports/ expression is closely correlated with a variety of biological processes and human diseases, such as chromosome dose compensation, development, tumorigenesis, and cardiovascular d iseases8–11. For example, Xu et al. reported that 172 lncRNAs and 188 mRNAs are differentially expressed in endometrial carcinoma tissues compared with normal tissues12. Several lncRNAs (KIAA0087, RP11-501O2, FAM212B-AS1, etc.) might be a key regulator of endometrial carcinogenesis and progression. Emerging evidence has also revealed the role of lncRNAs in the pathogenesis of EMs. A total of 576 differentially expressed lncRNAs were identified in the ectopic endometrium (ecEM) of adenomyosis, of which 388 lncRNAs were increased and 188 lncRNAs were d ecreased13. Zhang et al. demonstrated that the expression of lncRNA CCDC144NL-AS1 is upregulated in ectopic endometrial tissues compared with paired eutopic endometrial and normal endometrial (NE) t issues14. Functional studies further showed that CCDC144NL-AS1 inhibition decreases endometrial stromal cell (ESC) migration and i nvasion14. The role of lncRNA ADAMTS9-AS1 (hereafter named ADAMTS9-AS1) in tumor progression has been widely investigated. Low expression of ADAMTS9-AS1 predicts poorer survival than higher ADAMTS9-AS1 expression in patients with prostate cancer, and ADAMTS9-AS1 inhibits prostate cancer cell proliferation, indicating that ADAMTS9-AS1 functions as a tumor suppressor in prostate c ancer15. A recent study showed that ADAMTS9AS1 is significantly increased in ectopic endometrial t issues16. However, the biological role of ADAMTS9-AS1 in EMs remains unknown. Ferroptosis is an iron-dependent nonapoptotic type of cell death that distinctly differs from other forms of cell death, including apoptosis, pyroptosis, senescence and autophagy17–20. Overproduction of iron-induced lipid reactive oxygen species (ROS) is the crucial event in ferroptosis21,22. Although the levels of iron and lipid peroxide content are increased in the peritoneal fluid of women with EMs compared to those w ithout23–25, the role of ferroptosis in EMs has not been systematically investigated. In this study, we demonstrated that ADAMTS9AS1 was significantly upregulated in ecEM compared with eutopic endometrium (euEM). ADAMTS9-AS1 knockdown decreased cell viability and migration, whereas the effect was blocked by a ferroptosis inhibitor. ADAMTS9-AS1 functioned as a competing endogenous RNA (ceRNA) by sponging miR-6516-5p to derepress the expression of GPX4. Materials and methods A murine model of EMs. Animal experiments were approved by the Experimental Animal Committee of Shanghai Municipal Hospital of Traditional Chinese Medicine and carried out according to Shanghai Municipal Hospital of Traditional Chinese Medicine guidelines for the use of animals. All animal experiments were performed in accordance with the ARRIVE guidelines as described previously26 to minimize the number of animals used in the study and animal suffering. Female BALB/c mice (4–6 weeks old, 18–20 g) were obtained from Shanghai Regan Biotechnology Co., Ltd. (Shanghai, China) and were reared in a specific, pathogen-free facility27–29. After 1 week of acclimatization, mice were randomly divided into two groups: the donor group (n = 10) and recipient groups (n = 10). Ovariosteresis and estradiol valerate injection (0.5 µg/mouse/week; Aladdin, Shanghai, China) was carried out to avoid differences in the e (...truncated)