Upregulated Circular RNA hsa_circ_0008433 Regulates Pathogenesis in Endometriosis Via miRNA

circRNAs (circular RNAs) play important roles in the development of endometriosis. This study aimed to explore the functions of circRNAs on endometriosis. Two ectopic, two paired eutopic, and two normal endometrial tissue samples were collected for RNA-seq to obtain circRNA profiles and construct a circRNA–miRNA–mRNA network. The validation of 9 circRNAs in 15 patients was assessed by qRT-PCR. We selected hsa_circ_0008433 as the potential biomarker, followed by examining cell proliferation, colony formation, migration, angiopoiesis, cell cycle, and apoptosis. Furthermore, the expression of apoptosis-related proteins was detected using immunofluorescence (IF) and Western blotting. Bioinformatic analysis was used to select the potential target miRNA and genes of hsa_circ_0008433. A total of 209 upregulated and 117 downregulated differentially expressed circRNAs were identified from the eutopic and ectopic endometrial tissue samples. Eight circRNA levels were significantly increased in ectopic endometrial tissue sample compared with eutopic endometrial tissue. The hsa_circ_0008433 knockdown inhibited endometrial stromal cell proliferation, migration, colony formation, and angiopoiesis; promoted cell apoptosis; and downregulated Ki67 and PCNA expression levels. Moreover, the hsa_circ_0008433 knockdown increased Bax and E-CAD expression and decreased Bcl2, CDKN1B, and CyclinD1 levels. Ten potential target miRNAs of hsa_circ_0008433 were selected, and six of them occur significantly aberrant in hsa_circ_0008433-expressing cells. Increased hsa_circ_0008433 levels regulate epithelial mesenchymal transition (EMT) in endometriosis through the circRNA–miRNA–mRNA axis. Similar content being viewed by others Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Burney RO, Giudice LC. Pathogenesis and pathophysiology of endometriosis. Fertil Steril. 2012;98(3):511–9. https://doi.org/10.1016/j.fertnstert.2012.06.029. Soliman AM, Yang H, Du EX, Kelley C, Winkel C. The direct and indirect costs associated with endometriosis: a systematic literature review. Hum Reprod. 2016;31(4):712–22. https://doi.org/10.1093/humrep/dev335. Menakaya U, Infante F, Condous G. Consensus on current management of endometriosis. Hum Reprod. 2013;28(11):3162–3. https://doi.org/10.1093/humrep/det346. Wang K, Long B, Liu F, Wang JX, Liu CY, Zhao B, et al. A circular RNA protects the heart from pathological hypertrophy and heart failure by targeting miR-223. Eur Heart J. 2016;37(33):2602–11. https://doi.org/10.1093/eurheartj/ehv713. Hansen TB, Kjems J, Damgaard CK. Circular RNA and miR-7 in cancer. Cancer Res. 2013;73(18):5609–12. https://doi.org/10.1158/0008-5472.CAN-13-1568. Burd CE, Jeck WR, Liu Y, Sanoff HK, Wang Z, Sharpless NE. Expression of linear and novel circular forms of an INK4/ARF-associated non-coding RNA correlates with atherosclerosis risk. PLoS Genet. 2010;6(12):e1001233. https://doi.org/10.1371/journal.pgen.1001233. Floris G, Zhang L, Follesa P, Sun T. Regulatory role of circular RNAs and neurological disorders. Mol Neurobiol. 2017;54(7):5156–65. https://doi.org/10.1007/s12035-016-0055-4. Jeck WR, Sorrentino JA, Wang K, Slevin MK, Burd CE, Liu J, et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA. 2013;19(2):141–57. https://doi.org/10.1261/rna.035667.112. Qu S, Yang X, Li X, Wang J, Gao Y, Shang R, et al. Circular RNA: a new star of noncoding RNAs. Cancer Lett. 2015;365(2):141–8. https://doi.org/10.1016/j.canlet.2015.06.003. Zhang HM, Yang FQ, Chen SJ, Che J, Zheng JH. Upregulation of long non-coding RNA MALAT1 correlates with tumor progression and poor prognosis in clear cell renal cell carcinoma. Tumour Biol. 2015;36(4):2947–55. https://doi.org/10.1007/s13277-014-2925-6. Zhao ZJ, Shen J. Circular RNA participates in the carcinogenesis and the malignant behavior of cancer. RNA Biol. 2017;14(5):514–21. https://doi.org/10.1080/15476286.2015.1122162. Memczak S, Papavasileiou P, Peters O, Rajewsky N. Identification and characterization of circular RNAs as a new class of putative biomarkers in human blood. PLoS One. 2015;10(10):e0141214. https://doi.org/10.1371/journal.pone.0141214. Bahn JH, Zhang Q, Li F, Chan TM, Lin X, Kim Y, et al. The landscape of microRNA, Piwi-interacting RNA, and circular RNA in human saliva. Clin Chem. 2015;61(1):221–30. https://doi.org/10.1373/clinchem.2014.230433. Andre F, Arnedos M, Goubar A, Ghouadni A, Delaloge S. Ki67--no evidence for its use in node-positive breast cancer. Nat Rev Clin Oncol. 2015;12(5):296–301. https://doi.org/10.1038/nrclinonc.2015.46. Johnston SR, MacLennan KA, Sacks NP, Salter J, Smith IE, Dowsett M. Modulation of Bcl-2 and Ki-67 expression in oestrogen receptor-positive human breast cancer by tamoxifen. Eur J Cancer. 1994;30A(11):1663–9. Cabrera-Galeana P, Munoz-Montano W, Lara-Medina F, et al. Ki67 changes identify worse outcomes in residual breast cancer tumors after neoadjuvant chemotherapy. Oncologist. 2018;23(6):670–8. https://doi.org/10.1634/theoncologist.2017-0396. Frisa PS, Jacobberger JW. Cytometry of chromatin bound Mcm6 and PCNA identifies two states in G1 that are separated functionally by the G1 restriction point. BMC Cell Biol. 2010;11:26. https://doi.org/10.1186/1471-2121-11-26. De Craene B, Berx G. Regulatory networks defining EMT during cancer initiation and progression. Nat Rev Cancer. 2013;13(2):97–110. https://doi.org/10.1038/nrc3447. Basu A, Haldar S. The relationship between BcI2, Bax and p53: consequences for cell cycle progression and cell death. Mol Hum Reprod. 1998;4(12):1099–109. https://doi.org/10.1093/molehr/4.12.1099. Chang BL, Zheng SL, Isaacs SD, Wiley KE, Turner A, Li G, et al. A polymorphism in the CDKN1B gene is associated with increased risk of hereditary prostate cancer. Cancer Res. 2004;64(6):1997–9. Cheli Y, Giuliano S, Botton T, et al. Mitf is the key molecular switch between mouse or human melanoma initiating cells and their differentiated progeny. Oncogene. 2011;30(20):2307–18. https://doi.org/10.1038/onc.2010.598. Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell. 1991;65(4):701–13. Li Y, Zhang T, Qin S, Wang R, Li Y, Zhou Z, et al. Effects of UPF1 expression on EMT process by targeting Ecadherin, Ncadherin, Vimentin and Twist in a hepatocellular carcinoma cell line. Mol Med Rep. 2019;19(3):2137–43. https://doi.org/10.3892/mmr.2019.9838. Guo F, Cogdell D, Hu L, et al. MiR-101 suppresses the epithelial-to-mesenchymal transition by targeting ZEB1 and ZEB2 in ovarian carcinoma. Oncol Rep. 2014;31(5):2021–8. https://doi.org/10.3892/or.2014.3106. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281–97. Bentwich I, Avniel A, Karov Y, Aharonov R, Gilad S, Barad O, et al. Identification of hundreds of conserved and nonconserved human microRNAs. Nat Genet. 2005;37(7):766–70. https://doi.org/10.1038/ng1590. Park SM, Gaur AB, Lengyel E, Peter ME. The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev. 2008;22(7):894–907. https://doi.org/10.1101/gad.1640608. Korpal M, Lee ES, Hu G, Kang Y. The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2. J Biol Chem. 2008;283(22):14910–4. https://doi.org/10.1074/jbc.C800074200. Zhao Y, Samal E, Srivastava D. Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature. 2005;436(7048):214–20. https://doi.org/10.1038/nature03817. Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, Kirak O, et al. Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature. 2008;451(7182):1125–9. https://doi.org/10.1038/nature06607. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell. 2007;129(7):1401–14. https://doi.org/10.1016/j.cell.2007.04.040. Patterson EE, Holloway AK, Weng J, Fojo T, Kebebew E. MicroRNA profiling of adrenocortical tumors reveals miR-483 as a marker of malignancy. Cancer. 2011;117(8):1630–9. https://doi.org/10.1002/cncr.25724. Mayor-Lynn K, Toloubeydokhti T, Cruz AC, Chegini N. Expression profile of microRNAs and mRNAs in human placentas from pregnancies complicated by preeclampsia and preterm labor. Reprod Sci. 2011;18(1):46–56. https://doi.org/10.1177/1933719110374115. Qiao Y, Ma N, Wang X, Hui Y, Li F, Xiang Y, et al. MiR-483-5p controls angiogenesis in vitro and targets serum response factor. FEBS Lett. 2011;585(19):3095–100. https://doi.org/10.1016/j.febslet.2011.08.039. Wang J, Hou J, Zhang P, Li D, Zhang C, Liu J. Geniposide reduces inflammatory responses of oxygen-glucose deprived rat microglial cells via inhibition of the TLR4 signaling pathway. Neurochem Res. 2012;37(10):2235–48. Laudanski P, Charkiewicz R, Kuzmicki M, Szamatowicz J, Charkiewicz A, Niklinski J. MicroRNAs expression profiling of eutopic proliferative endometrium in women with ovarian endometriosis. Reprod Biol Endocrinol. 2013;11:78. https://doi.org/10.1186/1477-7827-11-78. Du XH, Shi G, Lu DH, et al. The expression of microRNA-221 in endometriosis and its impact on endometrial stromal cells. Sichuan Da Xue Xue Bao Yi Xue Ban. 2018;49(4):546–50. Li Y, Guessous F, Zhang Y, DiPierro C, Kefas B, Johnson E, et al. MicroRNA-34a inhibits glioblastoma growth by targeting multiple oncogenes. Cancer Res. 2009;69(19):7569–76. https://doi.org/10.1158/0008-5472.CAN-09-0529. Siemens H, Jackstadt R, Hunten S, et al. miR-34 and SNAIL form a double-negative feedback loop to regulate epithelial-mesenchymal transitions. Cell Cycle. 2011;10(24):4256–71. https://doi.org/10.4161/cc.10.24.18552. Menges CW, Kadariya Y, Altomare D, Talarchek J, Neumann-Domer E, Wu Y, et al. Tumor suppressor alterations cooperate to drive aggressive mesotheliomas with enriched cancer stem cells via a p53-miR-34a-c-Met axis. Cancer Res. 2014;74(4):1261–71. https://doi.org/10.1158/0008-5472.CAN-13-2062. Corney DC, Hwang CI, Matoso A, Vogt M, Flesken-Nikitin A, Godwin AK, et al. Frequent downregulation of miR-34 family in human ovarian cancers. Clin Cancer Res. 2010;16(4):1119–28. https://doi.org/10.1158/1078-0432.CCR-09-2642. Tazawa H, Tsuchiya N, Izumiya M, Nakagama H. Tumor-suppressive miR-34a induces senescence-like growth arrest through modulation of the E2F pathway in human colon cancer cells. Proc Natl Acad Sci U S A. 2007;104(39):15472–7. https://doi.org/10.1073/pnas.0707351104. Sun F, Fu H, Liu Q, Tie Y, Zhu J, Xing R, et al. Downregulation of CCND1 and CDK6 by miR-34a induces cell cycle arrest. FEBS Lett. 2008;582(10):1564–8. https://doi.org/10.1016/j.febslet.2008.03.057. Siemens H, Jackstadt R, Kaller M, Hermeking H. Repression of c-Kit by p53 is mediated by miR-34 and is associated with reduced chemoresistance, migration and stemness. Oncotarget. 2013;4(9):1399–415. https://doi.org/10.18632/oncotarget.1202. Urbich C, Kuehbacher A, Dimmeler S. Role of microRNAs in vascular diseases, inflammation, and angiogenesis. Cardiovasc Res. 2008;79(4):581–8. https://doi.org/10.1093/cvr/cvn156. Wu F, Yang Z, Li G. Role of specific microRNAs for endothelial function and angiogenesis. Biochem Biophys Res Commun. 2009;386(4):549–53. https://doi.org/10.1016/j.bbrc.2009.06.075. Ramon LA, Braza-Boils A, Gilabert-Estelles J, et al. MicroRNAs expression in endometriosis and their relation to angiogenic factors. Hum Reprod. 2011;26(5):1082–90. https://doi.org/10.1093/humrep/der025. Bou Kheir T, Futoma-Kazmierczak E, Jacobsen A, et al. miR-449 inhibits cell proliferation and is down-regulated in gastric cancer. Mol Cancer. 2011;10:29. https://doi.org/10.1186/1476-4598-10-29. Girardi C, De Pitta C, Casara S, et al. Integration analysis of microRNA and mRNA expression profiles in human peripheral blood lymphocytes cultured in modeled microgravity. Biomed Res Int. 2014;2014:296747–16. https://doi.org/10.1155/2014/296747. Geng Y, Zheng X, Hu W, Wang Q, Xu Y, He W, et al. Hsa_circ_0009361 acts as the sponge of miR-582 to suppress colorectal cancer progression by regulating APC2 expression. Clin Sci (Lond). 2019;133:1197–213. https://doi.org/10.1042/CS20190286. Zhang J, Hou L, Liang R, Chen X, Zhang R, Chen W, et al. CircDLST promotes the tumorigenesis and metastasis of gastric cancer by sponging miR-502-5p and activating the NRAS/MEK1/ERK1/2 signaling. Mol Cancer. 2019;18(1):80. https://doi.org/10.1186/s12943-019-1015-1. Mao W, Huang X, Wang L, Zhang Z, Liu M, Li Y, et al. Circular RNA hsa_circ_0068871 regulates FGFR3 expression and activates STAT3 by targeting miR-181a-5p to promote bladder cancer progression. J Exp Clin Cancer Res. 2019;38(1):169. https://doi.org/10.1186/s13046-019-1136-9. Code Availability Not applicable. Author information Authors and Affiliations Contributions HS conceived and designed the study, and critically revised the manuscript. NJ performed the experiments, analyzed the data, and drafted the manuscript. WP, JL, and TC participated in study design, study implementation, and manuscript revision. All authors read and approved the final manuscript. Corresponding author Ethics declarations Conflict of Interest The authors declare that they have no conflict of interest. Ethical Approval The study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-Sen University. Consent to Participate All the participants provided written informed consent before recruitment. Consent for Publication Patients signed informed consent regarding publishing their data and photographs. 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 circRNA cyclization sites of eight candidate circRNA verification by Sanger sequencing. Detection of eight candidate circRNAs using gel electrophoresis. (PNG 246 kb) Rights and permissions About this article Cite this article Jiang, N., Pan, W., Li, J. et al. Upregulated Circular RNA hsa_circ_0008433 Regulates Pathogenesis in Endometriosis Via miRNA. Reprod. Sci. 27, 2002–2017 (2020). https://doi.org/10.1007/s43032-020-00219-1 Received: Accepted: Published: Version of record: Issue date: DOI: https://doi.org/10.1007/s43032-020-00219-1