Homeobox C6 is Up-Regulated and Affects the Pathogenesis of Endometriosis

Endometriosis is one of the most common, difficult, and complicated gynecological disorders. The present study investigated the expression of HOXC6 in endometriosis and described its possible role in its pathogenesis. Ectopic and eutopic endometrial samples from 21 patients with endometriosis and a control endometrium from 15 women without endometriosis were collected. HOXC6 expression in endometrial tissue was analyzed with immunohistochemistry and quantitative reverse transcription polymerase chain reaction. HOXC6’s silencing in endometriotic stromal cells (ESC) was established with small interfering RNA (siRNA) to detect the effect on proliferation, adhension, invasion, and migration using CCK-8, adhesion, wound healing, and transwell assays. Western blotting was performed to detect the expression of related molecules after the down-regulation of HOXC6. HOXC6 mRNA and protein expression levels in the ectopic endometrial samples were significantly higher in women with endometriosis than in controls, while the levels in the eutopic endometrial tissues of the same patients did not significantly differ from those in non-endometriotic patients. The knockdown of HOXC6 expression inhibited the proliferation, adhesion, migration, and invasion of ESC. The results indicated that HOXC6 down-expression decreased the expression of N-cadherin and vimentin, whereas the expression of E-cadherin increased. HOXC6 down-expression also decreased the expression levels of TGF-β1 and phosphorylated SMAD2/SMAD3. In conclusion, HOXC6 was overexpressed in endometriosis and might therefore promote the proliferation, adhesion, invasion, and migration of ESC via the TGF-β1/smad signaling pathway. These findings present a new perspective and may therefore inspire further study of the endometriosis mechanism. Similar content being viewed by others Data Availability The datasets used or analyzed during the current study are available from the corresponding author on reasonable request. Abbreviations - ESC: - Endometriotic stromal cells - qRT-PCR: - Quantitative real-time polymerase chain reaction - siRNA: - Small interfering RNA - PBS: - Phosphate buffered saline - CCK: - Cell counting kit - NC: - Negative control - BC: - Blank control

Zondervan KT, Becker CM, Missmer SA. Endometriosis. N Engl J Med. 2020;382:1244–56. Saunders PTK, Horne AW. Endometriosis: Etiology, pathobiology, and therapeutic prospects. Cell. 2021;184:2807–24. Horne AW, Missmer SA. Pathophysiology, diagnosis, and management of endometriosis. BMJ. 2022;379:e070750. Stein S, Fritsch R, Lemaire L, Kessel M. Checklist: vertebrate homeobox genes. Mech Dev. 1996;55:91–108. Du H, Taylor HS. The role of HOX genes in female reproductive tract development, adult function, and fertility. Cold Spring Harb Perspect Med. 2015;6:a023002. Zanatta A, Rocha AM, Carvalho FM, Pereira RM, Taylor HS, Motta EL, et al. The role of the Hoxa10/HOXA10 gene in the etiology of endometriosis and its related infertility: a review. J Assist Reprod Genet. 2010;27:701–10. Esfandiari F, Favaedi R, Heidari-Khoei H, Chitsazian F, Yari S, Piryaei A, et al. Insight into epigenetics of human endometriosis organoids: DNA methylation analysis of HOX genes and their cofactors. Fertil Steril. 2021;115:125–37. Mihara Y, Maekawa R, Sato S, Shimizu N, Doi-Tanaka Y, Takagi H, et al. An integrated genomic approach identifies HOXC8 as an upstream regulator in ovarian endometrioma. J Clin Endocrinol Metab. 2020; 105(12). Ramachandran S, Liu P, Young AN, Yin-Goen Q, Lim SD, Laycock N, et al. Loss of HOXC6 expression induces apoptosis in prostate cancer cells. Oncogene. 2005;24(1):188–98. Fujiki K, Duerr EM, Kikuchi H, Ng A, Xavier RJ, Mizukami Y, et al. HOXC6 is overexpressed in gastrointestinal carcinoids and interacts with JunD to regulate tumor growth. Gastroenterology. 2008; 135(3): 907–916, 916 e901–902. Qi L, Chen J, Zhou B, Xu K, Wang K, Fang Z, et al. HomeoboxC6 promotes metastasis by orchestrating the DKK1/Wnt/β-catenin axis in right-sided colon cancer. Cell Death Dis. 2021;12:337. Eryi S, Zheng L, Honghua C, Su Z, Han X, Donggang P, et al. HOXC6 regulates the epithelial-mesenchymal transition through the TGF-β/Smad signaling pathway and predicts a poor prognosis in glioblastoma. J Oncol. 2022;2022:8016102. Li ZH, Ma Y, Zhou Y, Huang ZH. Expression profiles of HOXC6 predict the survival of glioblastoma patients and correlate with cell cycle. J Oncol. 2022;2022:8656865. Hamid AR, Hoogland AM, Smit F, Jannink S, van Rijt-van de Westerlo C, Jansen CF, et al. The role of HOXC6 in prostate cancer development. Prostate. 2015; 75: 1868–1876. McCabe CD, Spyropoulos DD, Martin D, Moreno CS. Genome-wide analysis of the homeobox C6 transcriptional network in prostate cancer. Cancer Res. 2008;68:1988–96. Li Q, Xi M, Shen F, Fu F, Wang J, Chen Y, et al. Identification of candidate gene signatures and regulatory networks in endometriosis and its related infertility by integrated analysis. Reprod Sci. 2022;29(2):411–26. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Am J Obstet Gynecol. 1975;122:262–3. Delbandi AA, Mahmoudi M, Shervin A, Akbari E, Jeddi-Tehrani M, Sankian M, et al. Eutopic and ectopic stromal cells from patients with endometriosis exhibit differential invasive, adhesive, and proliferative behavior. Fertil Steril. 2013;100(3):761–9. Zhao J, Lin H, Huang K, Li S. Cancer-associated fibroblasts-derived extracellular vesicles carrying lncRNA SNHG3 facilitate colorectal cancer cell proliferation via the miR-34b-5p/HuR/HOXC6 axis. Cell Death Discov. 2022;8:346. Zhou J, Yang X, Song P, Wang H, Wang X. HOXC6 in the prognosis of prostate cancer. Artif Cells Nanomed Biotechnol. 2019;47:2715–20. Li PD, Chen P, Peng X, Ma C, Zhang WJ, Dai XF. HOXC6 predicts invasion and poor survival in hepatocellular carcinoma by driving epithelial-mesenchymal transition. Aging (Albany NY). 2018;10:115–30. Dongre A, Weinberg RA. New insights into the mechanisms of epithelial-mesenchymal transition and implications for cancer. Nat Rev Mol Cell Biol. 2019;20:69–84. Konrad L, Dietze R, Riaz MA, Scheiner-Bobis G, Behnke J, Horné F, et al. Epithelial-mesenchymal transition in endometriosis-when does it happen? J Clin Med. 2020;9:1915. Zhang M, Xu T, Tong D, Li S, Yu X, Liu B, et al. Research advances in endometriosis-related signaling pathways: A review. Biomed Pharmacother. 2023;164:114909. Gao T, Cao Y, Hu M, Du Y. The activation of TGF-β signaling promotes cell migration and invasion of ectopic endometrium by targeting NRP2. Reprod Biol. 2022;22:100697. Zhang J, Li H, Yi D, Lai C, Wang H, Zou W, et al. Knockdown of vascular cell adhesion molecule 1 impedes transforming growth factor beta 1-mediated proliferation, migration, and invasion of endometriotic cyst stromal cells. Reprod Biol Endocrinol. 2019;17:69. Shi LB, Zhou F, Zhu HY, Huang D, Jin XY, Li C, et al. Transforming growth factor beta1 from endometriomas promotes fibrosis in surrounding ovarian tissues via Smad2/3 signaling. Biol Reprod. 2017;97:873–82. Zhang F, Ren CC, Liu L, Chen YN, Yang L, Zhang XA. HOXC6 gene silencing inhibits epithelial-mesenchymal transition and cell viability through the TGF-β/smad signaling pathway in cervical carcinoma cells. Cancer Cell Int. 2018;18:204. Funding The Wisdom Accumulation and Talent Cultivation Project of the Third Xiangya Hospital of Central South University [No. YX202112]. Author information Authors and Affiliations Contributions JFJ conducted the experiments and drafted the manuscript. LYZ contributed to tissue sample collection. LJL contributed to project design and revised the manuscript. All authors read and approved the final manuscript. Corresponding author Ethics declarations Ethics Approval The study protocol was approved by the institutional review board of the Third Xiangya Hospital of Central South University (No.E22250), and informed consent was obtained from all the patients. Conflict of interest The authors declare that they have no conflict of interest. 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. Rights and permissions Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. About this article Cite this article Jiang, J., Zhang, L. & Li, L. Homeobox C6 is Up-Regulated and Affects the Pathogenesis of Endometriosis. Reprod. Sci. 32, 2574–2582 (2025). https://doi.org/10.1007/s43032-025-01918-3 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-025-01918-3