Linc-ROR Promotes EMT by Targeting miR-204-5p/SMAD4 in Endometriosis

Endometriosis (EMs) is a systemic and chronic disease with cancer-like feature, namely, distant implantation, which caused heavy healthy burden of nearly 200 million females. LncRNAs have been proved as new modulators in epithelial–mesenchymal transition (EMT) and EMs. Quantitative real-time PCR was conducted to measure the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (Linc-ROR), and miR-204-5p in ectopic endometrium (n = 25), eutopic endometrium (n = 20), and natural control endometrium (n = 22). Overexpression of Linc-ROR, knockdown or overexpression of miR-204-5p in End1/E6E7 and Ishikawa cells, was conducted to detect the function of Linc-ROR and miR-204-5p in EMs. Furthermore, luciferase reports were used to confirm the combination of Linc-ROR and miR-204-5p and the combination between miR-204-5p and SMAD4. Cell-Counting Kit-8, EdU assay, transwell assays, and Western blotting were used to detect the function of Linc-ROR and miR-204-5p in EMs cancer-like behaviors and EMT process. Linc-ROR was up-regulated in ectopic endometrium. Overexpressed Linc-ROR promotes cell proliferation, invasion, and EMT process. Linc-ROR regulated the EMT process, cellular proliferation, and invasion of EMs via binding to miR-204-5p. In addition, overexpression of Linc-ROR up-regulated SMAD4, a target protein of miR-204-5p, with which regulated EMT process and cancer-like behaviors in EMs together. Linc-ROR/miR-204-5p/SMAD4 axis plays a vital role in regulation EMT process in EMs, which might become a novel therapeutic targets and powerful biomarkers in EMs therapy. Similar content being viewed by others Data Availability Not applicable. Code Availability Not applicable. Abbreviations - EMs: - Endometriosis - EMT: - Epithelial–mesenchymal transition - ncRNA: - Non-coding RNA - ISK: - Ishikawa - E6E7: - End1/E6E7 - E-cad: - E-cadherin - VIM: - Vimentin - ceRNA: - Competing endogenous RNA - LncRNA: - Long non-coding RNA - PBS: - Phosphate-buffered saline - CCK-8: - Cell counting kit-8 - qRT-PCR: - Quantitative reverse-transcription polymerase chain reaction - IHC: - Immunohistochemistry - EC: - Ectopic endometrium - EU: - Eutopic endometrium - NC: - Endometrium from the control group - R-SMAD: - Receptor-regulated SMAD

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The authors thank AiMi Academic Services (www.aimieditor.com) for the English language editing and review services. Funding This work was supported by the National Natural Science Foundation of China (81671437, 81771558) and the Natural Science Foundation of Hunan Province, China (2016JC2049, 2020JJ4814). Author information Authors and Affiliations Contributions Mingyu Yi: Data curation, methodology, software, writing—original draft preparation. Sixue Wang: Data curation, Writing—Original draft preparation. Xinyue Zhang: Data curation, writing—original draft preparation. Li Jiang: Resources, Investigation, validation. Xiaomeng Xia: Resources, investigation, validation. Tingting Zhang: Resources, investigation, validation. Xiaoling Fang: Supervision, writing—reviewing and editing, funding acquisition. Corresponding author Ethics declarations Ethics Approval This study was approved by the Institutional Ethics Review Committee of Second Xiangya Hospital of Central South University (#2016243). Consent to Participate Not applicable. Consent for Publication Not applicable. Competing Interests The authors declare no competing interests. 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. Fig. S1 (download PNG ) Validation of transfection plasmid Linc-ROR, miR-204-5p mimic and miR-204-5p Inhibitor. a Plasmid map of Linc-ROR. b 1 μg plasmid Linc-ROR and vector was transfected into End1/E6E7 and Ishikawa cells respectively, and qRT-PCR was conducted to measure the relatively expression of Linc-ROR. Two-tailed Student’s t-test was conducted for statistical analysis. c 50 nM miR-204-5p mimic or mimic NC was transfected into End1/E6E7 and Ishikawa cells, 80 nM miR-204-5p Inhibitor or Inhibitor NC was transfected into Ishikawa cells, and 30 nM miR-204-5p Inhibitor or Inhibitor NC was transfected into End1/E6E7 cells, the transfection was confirmed using qRT-PCR. Two-tailed Student’s t-test was conducted for statistical analysis. E6E7, End1/E6E7; ISK, Ishikawa; mimic, miR-204-5p mimic; inhibitor, miR-204-5p Inhibitor; NC, natural control. (PNG 214 kb) Fig. S2 (download PNG ) Expression of miR-124-3p, miR-138-5p, miR-195-5p and miR-205-5p, and their regulation by Linc-ROR. a-d Left panel shows expression levels of miR-124-3p, miR-138-5p, miR-195-5p and miR-205-5p in ectopic endometrium (EC, n = 10), eutopic endometrium (EU, n = 10), and control group (NC, n = 10) by qRT-PCR. Kruskal–Wallis test followed by pot-hoc Dunn’s test was conducted for statistical analysis. Right panel shows expression of miR-124-3p, miR-138-5p, miR-195-5p and miR-205-5p in End1/E6E7 and Ishikawa cells transfected with Linc-ROR plasmid or control vector using qRT-PCR. Two-tailed Student’s t-test was conducted for statistical analysis. E6E7, End1/E6E7; ISK, Ishikawa. (PNG 236 kb) 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 Yi, M., Wang, S., Zhang, X. et al. Linc-ROR Promotes EMT by Targeting miR-204-5p/SMAD4 in Endometriosis. Reprod. Sci. 30, 2665–2679 (2023). https://doi.org/10.1007/s43032-023-01204-0 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-023-01204-0