Dysregulation of miR-202-3p Affects Migration and Invasion of Endometrial Stromal Cells in Endometriosis via Targeting ROCK1

Dysregulation of microRNAs in endometrial cells plays a pivotal role in the pathogenesis of endometriosis (EM). This study aims to investigate the implication of aberrant miR-202-3p expression in EM and the underlying mechanisms. We demonstrated that miR-202-3p was significantly downregulated in eutopic endometrium of EM in comparison to normal endometrial samples (P < 0.05). Primary endometrial stromal cells (ESCs) isolated from eutopic or ectopic endometrium also showed a significant decrease in miR-202-3p level compared to ESCs from normal endometrium (P < 0.01). Functional studies using MTT, wound healing assay and transwell assay indicated that overexpression of miR-202-3p greatly impaired cell viability, migration, and invasion, whereas suppression of miR-202-3p exhibited the opposite effects (P < 0.05 or P < 0.01). miR-202-3p mimics or inhibitors transfection significantly decreased or increased expression of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1), respectively, in ESCs (P < 0.01). Using dual luciferase reporter assay, we validated ROCK1 as a direct target of miR-202-3p. Moreover, negative correlations between miR-202-3p and ROCK1 mRNA/protein levels were determined in both eutopic and normal control endometrium (P < 0.01). In conclusion, these findings suggest that suppression of miR-202-3p in ESCs results in enhanced cell viability, invasion, and migration at least partially via upregulation of its target ROCK1, which eventually contributes to the development of endometriosis. Similar content being viewed by others Change history 25 April 2022 A Correction to this paper has been published: https://doi.org/10.1007/s43032-022-00950-x

Giudice LC, Kao LC. Endometriosis. Lancet. 2004;364(9447):1789–99. Vercellini P, Vigano P, Somigliana E, Fedele L. Endometriosis: pathogenesis and treatment. Nat Rev Endocrinol. May 2014;10(5):261–75. Rutherford EJ, Hill ADK, Hopkins AM. Adhesion in physiological, benign and malignant proliferative states of the endometrium: microenvironment and the clinical big picture. Cells. 2018;7(5). Soni UK, Chadchan SB, Kumar V, Ubba V, Khan MTA, Vinod BSV, et al. A high level of TGF-B1 promotes endometriosis development via cell migration, adhesiveness, colonization, and invasiveness. Biol Reprod. 2019;100(4):917–38. Laschke MW, Menger MD. Basic mechanisms of vascularization in endometriosis and their clinical implications. Human reproduction update. Jan 25 2018. Liu H, Lang JH. Is abnormal eutopic endometrium the cause of endometriosis? The role of eutopic endometrium in pathogenesis of endometriosis. Med Sci Monit. 2011;17(4):Ra92–9. Sundqvist J, Andersson KL, Scarselli G, Gemzell-Danielsson K, Lalitkumar PG. Expression of adhesion, attachment and invasion markers in eutopic and ectopic endometrium: a link to the aetiology of endometriosis. Hum Reprod. Sep 2012;27(9):2737–46. Jiang QY, Xia JM, Ding HG, Fei XW, Lin J, Wu RJ. RNAi-mediated blocking of ezrin reduces migration of ectopic endometrial cells in endometriosis. Mol Hum Reprod. 2012;18(9):435–41. Chen Q, Hang Y, Zhang T, et al. USP10 promotes proliferation and migration and inhibits apoptosis of endometrial stromal cells in endometriosis through activating the Raf-1/MEK/ERK pathway. Am J Physiol Cell Physiol. 2018;315(6):C863–c872. Ohlsson Teague EM, Van der Hoek KH, Van der Hoek MB, et al. MicroRNA-regulated pathways associated with endometriosis. Mol Endocrinol. Feb 2009;23(2):265–75. Panir K, Schjenken JE, Robertson SA, Hull ML. Non-coding RNAs in endometriosis: a narrative review. Human reproduction update. Jul 1 2018;24(4):497–515. Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215–33. Ambros V. The functions of animal microRNAs. Nature. 2004;431(7006):350–5. Li SC, Tang P, Lin WC. Intronic microRNA: discovery and biological implications. DNA Cell Biol. 2007;26(4):195–207. Hull ML, Nisenblat V. Tissue and circulating microRNA influence reproductive function in endometrial disease. Reprod BioMed Online. 2013;27(5):515–29. Santamaria X, Taylor H. MicroRNA and gynecological reproductive diseases. Fertil Steril. 2014;101(6):1545–51. Braza-Boils A, Mari-Alexandre J, Gilabert J, et al. MicroRNA expression profile in endometriosis: its relation to angiogenesis and fibrinolytic factors. Hum Reprod. 2014;29(5):978–88. Hoffman AE, Liu R, Fu A, et al. Targetome profiling, pathway analysis and genetic association study implicate miR-202 in lymphomagenesis. Cancer Epidemiol Biomark Prev. Mar 2013;22(3):327–36. Laird SM, Widdowson R, El-Sheikhi M, et al. Expression of CXCL12 and CXCR4 in human endometrium; effects of CXCL12 on MMP production by human endometrial cells. Hum Reprod (Oxford, England). 2011;26(5):1144–52. Ng EK, Chong WW, Jin H, Lam EK, Shin VY, Yu J, et al. Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening. Gut. 2009;58(10):1375–81. Halme J, Hammond MG, Hulka JF, Raj SG, Talbert LM. Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol. 1984;64(2):151–4. Colón-Caraballo M, Torres-Reverón A, Soto-Vargas JL, Young SL, Lessey B, Mendoza A, et al. Effects of histone methyltransferase inhibition in endometriosis. Biol Reprod. 2018;99(2):293–307. Liu H, Zhang Z, Xiong W, Zhang L, Xiong Y, Li N, et al. Hypoxia-inducible factor-1α promotes endometrial stromal cells migration and invasion by upregulating autophagy in endometriosis. Reproduction. 2017;153(6):809–20. Nothnick WB. The role of micro-RNAs in the female reproductive tract. Reproduction. 2012;143(5):559–76. Burney RO, Hamilton AE, Aghajanova L, Vo KC, Nezhat CN, Lessey BA, et al. MicroRNA expression profiling of eutopic secretory endometrium in women with versus without endometriosis. Mol Hum Reprod. 2009;15(10):625–31. Pan Q, Luo X, Toloubeydokhti T, Chegini N. The expression profile of micro-RNA in endometrium and endometriosis and the influence of ovarian steroids on their expression. Mol Hum Reprod. 2007;13(11):797–806. Nematian SE, Mamillapalli R, Kadakia TS, et al. Systemic Inflammation Induced by microRNAs: endometriosis-derived alterations in circulating microRNA 125b-5p and Let-7b-5p regulate macrophage cytokine production. J Clin Endocrinol Metab. 2018;103(1):64–74. Ma Y, Huang YX, Chen YY. miRNA-34a-5p downregulation of VEGFA in endometrial stem cells contributes to the pathogenesis of endometriosis. Mol Med Rep. 2017;16(6):8259–64. Liang Z, Chen Y, Zhao Y, et al. miR-200c suppresses endometriosis by targeting MALAT1 in vitro and in vivo. 2017;8(1):251. Peiffer SL, Herzog TJ, Tribune DJ, Mutch DG, Gersell DJ, Goodfellow PJ. Allelic loss of sequences from the long arm of chromosome 10 and replication errors in endometrial cancers. Cancer Res. 1995;55(9):1922–6. Han X, Wang Q, Wang Y, et al. Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1/microRNA-202-3p/periostin axis modulates invasion and epithelial-mesenchymal transition in human cervical cancer. J Cell Physiol. 2019. Wang Q, Huang Z, Guo W, et al. microRNA-202-3p inhibits cell proliferation by targeting ADP-ribosylation factor-like 5A in human colorectal carcinoma. Clin Cancer Res. 2014;20(5):1146–57. Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 2007;129(7):1401–14. Sontakke SD, Mohammed BT, McNeilly AS, Donadeu FX. Characterization of microRNAs differentially expressed during bovine follicle development. Reproduction (Cambridge, England). 2014;148(3):271–83. Arnold JT, Kaufman DG, Seppala M, Lessey BA. Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model. Hum Reprod. 2001;16(5):836–45. Yang C, Yao C, Tian R, et al. miR-202-3p regulates Sertoli cell proliferation, synthesis function, and apoptosis by targeting LRP6 and cyclin D1 of Wnt/beta-Catenin signaling. Mol Ther Nucleic Acids. 2019;14:1–19. Zhao Y, Li C, Wang M, Su L, Qu Y, Li J, et al. Decrease of miR-202-3p expression, a novel tumor suppressor, in gastric cancer. PLoS One. 2013;8(7):e69756. Diao CY, Guo HB, Ouyang YR, Zhang HC, Liu LH, Bu J, et al. Screening for metastatic osteosarcoma biomarkers with a DNA microarray. Asian Pac J Cancer Prev. 2014;15(4):1817–22. Meola J, Hidalgo Gdos S, Silva JC, et al. Caldesmon: new insights for diagnosing endometriosis. Biol Reprod. 2013;88(5):122. He Y, Guo Q, Cheng Y, et al. Abnormal activation of the sonic hedgehog signaling pathway in endometriosis and its diagnostic potency. Fertil Steril. 2018;110(1):128–136.e2. Heard ME, Simmons CD, Simmen FA, Simmen RC. Krüppel-like factor 9 deficiency in uterine endometrial cells promotes ectopic lesion establishment associated with activated notch and hedgehog signaling in a mouse model of endometriosis. Endocrinology. 2014;155(4):1532–46. Ping S, Ma C, Liu P, Yang L, Yang X, Wu Q, et al. Molecular mechanisms underlying endometriosis pathogenesis revealed by bioinformatics analysis of microarray data. Arch Gynecol Obstet. 2016;293(4):797–804. Hirakawa T, Nasu K, Abe W, Aoyagi Y, Okamoto M, Kai K, et al. miR-503, a microRNA epigenetically repressed in endometriosis, induces apoptosis and cell-cycle arrest and inhibits cell proliferation, angiogenesis, and contractility of human ovarian endometriotic stromal cells. Hum Reprod. 2016;31(11):2587–97. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature. 2002;420(6916):629–35. Guo D, Li Y, Chen Y, Zhang D, Wang X, Lu G, et al. DANCR promotes HCC progression and regulates EMT by sponging miR-27a-3p via ROCK1/LIMK1/COFILIN1 pathway. Cell Prolif. 2019;52(4):e12628. Di Pietro C, Caruso S, Battaglia R, et al. MiR-27a-3p and miR-124-3p, upregulated in endometrium and serum from women affected by chronic Endometritis, are new potential molecular markers of endometrial receptivity. Am J Reprod Immunol. 2018;80(3):e12858. Kang CG, Han HJ, Lee HJ, Kim SH, Lee EO. Rho-associated kinase signaling is required for osteopontin-induced cell invasion through inactivating cofilin in human non-small cell lung cancer cell lines. Bioorg Med Chem Lett. 2015;25(9):1956–60. Yin J, Lv L, Zhai P, Long T, Zhou Q, Pan H, et al. Connexin 40 regulates lung endothelial permeability in acute lung injury via the ROCK1-MYPT1- MLC20 pathway. Am J Physiol Lung Cell Mol Physiol. 2019;316(1):L35–44. Zhang Z, Chen P, Guo C, Meng X, Wang D. Effect of LIM kinase 1 overexpression on behaviour of endometriosis-derived stromal cells. Cell Tissue Res. 2015;359(3):885–93. Yotova I, Quan P, Gaba A, Leditznig N, Pateisky P, Kurz C, et al. Raf-1 levels determine the migration rate of primary endometrial stromal cells of patients with endometriosis. J Cell Mol Med. 2012;16(9):2127–39. Gong J, Guan L, Tian P, Li C, Zhang Y. Rho kinase type 1 (ROCK1) promotes lipopolysaccharide-induced inflammation in corneal epithelial cells by activating toll-like receptor 4 (TLR4)-mediated signaling. Med Sci Monit. 2018;24:3514–23. Yun BH, Chon SJ, Choi YS, et al. Pathophysiology of endometriosis: role of high mobility group box-1 and toll-like receptor 4 developing inflammation in endometrium. PLoS One. 2016;11(2):e0148165. Azuma Y, Taniguchi F, Nakamura K, et al. Lipopolysaccharide promotes the development of murine endometriosis-like lesions via the nuclear factor-kappa B pathway. Am J Reprod Immunol. 2017;77(4). Bryan BA, Dennstedt E, Mitchell DC, Walshe TE, Noma K, Loureiro R, et al. RhoA/ROCK signaling is essential for multiple aspects of VEGF-mediated angiogenesis. FASEB J. 2010;24(9):3186–95. Suarez Y, Fernandez-Hernando C, Yu J, et al. Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis. Proc Natl Acad Sci USA. 2008;105(37):14082–7. Wang S, Olson EN. AngiomiRs--key regulators of angiogenesis. Curr Opin Genet Dev. 2009;19(3):205–11. Akoum A, Al-Akoum M, Lemay A, et al. Imbalance in the peritoneal levels of interleukin 1 and its decoy inhibitory receptor type II in endometriosis women with infertility and pelvic pain. Fertil Steril. 2008;89(6):1618–24. Shi C, Wu L, Lin W, Cai Y, Zhang Y, Hu B, et al. MiR-202-3p regulates interleukin-1β-induced expression of matrix metalloproteinase 1 in human nucleus pulposus. Gene. 2019;687:156–65. Acknowledgments The authors thank the support for this research from National Natural Science Foundation of China (NSFC) (No. 81100411 to M.Z.), Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund (No. cxpy20160038 to M.Z.), Hubei Province health and family planning scientific research project (No. WJ2018H0007 to M.Z.), and Health Commission of Hubei Province scientific research project (No. WJ2019Q048 to M.Z.) Funding The present study was supported by National Natural Science Foundation of China (NSFC) (No. 81100411 to M.Z.), Zhongnan Hospital of Wuhan University Science, Technology and Innovation Seed Fund (No. cxpy20160038 to M.Z.), Hubei Province health and family planning scientific research project (No. WJ2018H0007 to M.Z.), Health Commission of Hubei Province scientific research project (No. WJ2019Q048 to M.Z.) Author information Authors and Affiliations Contributions MZ performed most of the experiments and contributed to study design and manuscript drafting; YZ revised the manuscript, LL was responsible for data analysis, LM and CZ provided aid with collection and processing of samples. All authors reviewed and approved the final manuscript. Corresponding author Ethics declarations Conflict of Interest The authors declare that they have no conflicts of interest. Additional information Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Electronic supplementary material ESM 1 (download DOCX ) (DOCX 1241 kb) Rights and permissions About this article Cite this article Zhang, M., Zhang, Y., Li, L. et al. Dysregulation of miR-202-3p Affects Migration and Invasion of Endometrial Stromal Cells in Endometriosis via Targeting ROCK1. Reprod. Sci. 27, 731–742 (2020). https://doi.org/10.1007/s43032-019-00079-4 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-019-00079-4