Abstract
It is suggested that aberrantly expressed microRNAs are involved in the pathogenesis of endometriosis. Our previous study demonstrated that expression of the microRNA hsa-miR-199a-3p is attenuated in human endometriotic cyst stromal cells (ECSCs). The current study aimed to define the roles of hsa-miR-199a-3p in the development of endometriosis. ECSCs and normal endometrial stromal cells (NESCs) were isolated from ovarian endometrioma and normal endometrial tissues, respectively. We evaluated the effect of transfected hsa-miR-199a-3p on the migration, invasion, and contractility of ECSCs using Transwell migration assays, in vitro wound healing assays, Transwell invasion assays, and collagen gel contraction assays. We also examined the downstream target of hsa-miR-199a-3p with an online public database search and luciferase reporter assay. Expression of hsa-miR-199a-3p in ECSCs was significantly lower than that in NESCs, whereas the expression of p21-activated kinase 4 (PAK4) mRNA was significantly higher. Transfection of hsa-miR-199a-3p inhibited the migration, invasion, and contractility of ECSCs via inhibition of PAK4 mRNA expression. PAK4 was confirmed to be the direct target of hsa-miR-199a-3p. Transfection of PAK4 small interfering RNA and the PAK4 inhibitor PF-3758309 also inhibited ECSC migration, invasion, and contractility. These findings suggest that hsa-miR-199a-3p may act as a tumor suppressor in endometriosis development. Attenuation of hsa-miR-199a-3p expression was favorable for ECSCs to acquire the highly invasive, motile, and contractile characteristics of endometriotic cells and is involved in the development of endometriosis. Accordingly, PAK4 inhibitors may be promising for the treatment of endometriosis.
Similar content being viewed by others
References
Giudice LC. Clinical practice. Endometriosis N Engl J Med. 2010;362:2389–98.
Pearce CL, Templeman C, Rossing MA, Lee A, Near AM, Webb PM, et al. Ovarian Cancer Association Consortium. Association between endometriosis and risk of histological subtypes of ovarian cancer: a pooled analysis of case-control studies. Lancet Oncol. 2012;13:385–94.
Abe W, Nasu K, Nakada C, Kawano Y, Moriyama M, Narahara H. miR-196b targets c-Myc and Bcl-2 expression, inhibits proliferation and induces apoptosis in endometriotic stromal cells. Hum Reprod. 2013;28:750–61.
Okamoto M, Nasu K, Abe W, Aoyagi Y, Kawano Y, Kai K, et al. Enhanced miR-210 expression promotes the pathogenesis of endometriosis through activation of signal transducer and activator of transcription 3. Hum Reprod. 2015;30:632–41.
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;31:2587–97.
Aoyagi Y, Nasu K, Kai K, Hirakawa T, Okamoto M, Kawano Y, et al. Decidualization differentially regulates microRNA expression in eutopic and ectopic endometrial stromal cells. Reprod Sci. 2017;24:445–55.
Takebayashi K, Nasu K, Okamoto M, Aoyagi Y, Hirakawa T, Narahara H. hsa-miR-100-5p, an overexpressed miRNA in human ovarian endometriotic stromal cells, promotes invasion through attenuation of SMARCD1 expression. Reprod Biol Endocrinol. 2020;18:31.
Henry JC, Park J-K, Jiang J, Kim JH, Roberts LR, Banerjee S, et al. miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines. Biochem Biophys Res Commun. 2010;403:120–5.
Hou J, Lin L, Zhou W, Wang Z, Ding G, Dong Q, et al. Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma. Cancer Cell. 2011;19:232–43.
Minna E, Romeo P, De Cecco L, Dugo M, Cassinelli G, Pilotti S, et al. miR-199a-3p displays tumor suppressor functions in papillary thyroid carcinoma. Oncotarget. 2014;5:2513–28.
Wang S-H, Zhou J-D, He Q-Y, Yin Z-Q, Cao K, Luo C-Q. MiR-199a inhibits the ability of proliferation and migration by regulating CD44-Ezrin signaling in cutaneous squamous cell carcinoma cells. Int J Clin Exp Pathol. 2014;7:7131–41.
Wu D, Huang HJ, He CN, Wang KY. MicroRNA-199a-3p regulates endometrial cancer cell proliferation by targeting mammalian target of rapamycin (mTOR). Int J Gynecol Cancer. 2013;23:1191–7.
Tsukigi M, Bilim V, Yuuki K, Ugolkov A, Naito S, Nagaoka A, et al. Re-expression of miR-199a suppresses renal cancer cell proliferation and survival by targeting GSK-3beta. Cancer Lett. 2012;315:189–97.
Liu XJ, Bai XG, Teng YL, Song L, Lu N, Yang RQ. miRNA-15a-5p regulates VEGFA in endometrial mesenchymal stem cells and contributes to the pathogenesis of endometriosis. Eur Rev Med Pharmacol Sci. 2016;20:3319–26.
Duan Z, Choy E, Harmon D, Liu X, Susa M, Mankin H, et al. MicroRNA-199a-3p is downregulated in human osteosarcoma and regulates cell proliferation and migration. Mol Cancer Ther. 2011;10:1337–45.
Wang Z, Ting Z, Li Y, Chen G, Lu Y, Hao X. microRNA-199a is able to reverse cisplatin resistance in human ovarian cancer cells through the inhibition of mammalian target of rapamycin. Oncol Lett. 2013;6:789–94.
Li S-Q, Wang Z-H, Mi X-G, Liu L, Tan Y. miR-199a-3p suppresses migration and invasion of breast cancer cells by downregulating PAK4/MEK/ERK signaling pathway. Int Union Biochem Mol Biol Life. 2015;67:768–77.
Qin Z, Wei X, Jin N, Wang Y, Zhao R, Hu Y, et al. MiR-199a targeting ROCK1 to affect kidney cell proliferation, invasion and apoptosis. Artif Cells Nanomed Biotechnol. 2018;46:1920–5.
Ecke TH, Stier K, Weickmann S, Zhao Z, Buckendahl L, Stephan C, et al. miR-199a-3p and miR-214-3p improve the overall survival prediction of muscle-invasive bladder cancer patients after radical cystectomy. Cancer Med. 2017;6:2252–62.
Qu Y, Huang X, Li Z, Liu J, Wu J, Chen D, et al. miR-199a-3p inhibits aurora kinase A and attenuates prostate cancer growth. New avenue for prostate cancer treatment. Am J Pathol. 2014;184:1541–9.
Desjobert C, Carrier A, Delmas A, Marzese DM, Daunay A, Busato F, et al. Demethylation by low-dose 5-aza-2′-deoxycytidine impairs 3D melanoma invasion partially through miR-199a-3p expression revealing the role of this miR in melanoma. Clin Epigenetics. 2019;11:9.
Jiang J, Gusev Y, Aderca I, Mettler TA, Nagorney DM, Brackett DJ, et al. Association of microRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival. Clin Cancer Res. 2008;14:419–27.
Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin SF, Dunning MJ, et al. MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biol. 2007;8:R214.
Nishida M, Nasu K, Fukuda J, Kawano Y, Narahara H, Miyakawa I. Down regulation of interleukin-1 receptor expression causes the dysregulated expression of CXC chemokines in endometriotic stromal cells: a possible mechanism for the altered immunological functions in endometriosis. J Clin Endocrinol Metab. 2004;89:5094–100.
Nasu K, Nishida M, Ueda T, Takai N, Sun B, Narahara H, et al. Bufalin induces apoptosis and the G0/G1 cell cycle arrest of endometriotic stromal cells: a promising agent for the treatment of endometriosis. Mol Hum Reprod. 2005;11:817–23.
Matsumoto H, Nasu K, Nishida M, Ito H, Bing S, Miyakawa I. Regulation of proliferation, motility, and contractility of human endometrial stromal cells by platelet-derived growth factor. J Clin Endocrinol Metab. 2005;90:3560–7.
Nasu K, Nishida M, Matsumoto H, Sun B, Inoue C, Kawano Y, et al. Regulation of proliferation, motility, and contractivity of cultured human endometrial stromal cells by transforming growth factor-beta isoforms. Fertil Steril. 2005;84(Suppl):1114–23.
Tsuno A, Nasu K, Kawano Y, Yuge A, Li H, Abe W, et al. Fasudil inhibits the proliferation and contractility and induces cell cycle arrest and apoptosis of human endometriotic stromal cells: a promising agent for the treatment of endometriosis. J Clin Endocrinol Metab. 2011;96:E1944–52.
Phatak P, Burrows WM, Chesnick IE, Tulapurkar ME, Rao JN, Turner DJ, et al. MiR-199a-3p decreases esophageal cancer cell proliferation by targeting p21 activated kinase 4. Oncotarget. 2018;9:28391–407.
Zeng B, Shi W, Tan G. MiR-199a/b-3p inhibits gastric cancer cell proliferation via down-regulating PAK4/MEK/ERK signaling pathway. BMC Cancer. 2018;18:34.
Dai L, Gu L, Di W. MiR-199a attenuates endometrial stromal cell invasiveness through suppression of the IKKβ/NF-κB pathway and reduced interleukin-8 expression. Mol Hum Reprod. 2012;18:136–45.
Dart AE, Wells CM. P21-activated kinase 4--not just one of the PAK. Eur J Cell Biol. 2013;92:129–38.
King H, Nicholas NS, Wells CM. Role of p-21-activated kinases in cancer progression. Int Rev Cell Mol Biol. 2014;309:347.
Rane CK, Minden A. P21 activated kinase signaling in cancer. Semin Cancer Biol. 2019;54:40–9.
Siu MK, Chan HY, Kong DSH, Wong ESY, Wong OGW, Ngan HYS, et al. p21-activated kinase 4 regulates ovarian cancer cell proliferation, migration, and invasion and contributes to poor prognosis in patients. Proc Natl Acad Sci U S A. 2010;107:18622–7.
Ahn HK, Jang J, Lee J, Park SH, Park JO, Park YS, et al. P21-activated kinase 4 overexpression in metastatic gastric cancer patients. Transl Oncol. 2011;4:345–9.
Minden A. The pak4 protein kinase in breast cancer. ISRN Oncol. 2012;2012:694201.
Park MH, Lee HS, Lee CS, You ST, Kim DJ, Park BH, et al. p21-Activated kinase 4 promotes prostate cancer progression through CREB. Oncogene. 2013;32:2475–82.
Wong LE, Chen N, Karantza V, Minden A. The Pak4 protein kinase is required for oncogenic transformation of MDA-MB-231 breast cancer cells. Oncogenesis. 2013;2:e50.
Tyagi N, Bhardwaj A, Singh AP, McClellan S, Carter JE, Singh S. p-21 activated kinase 4 promotes proliferation and survival of pancreatic cancer cells through AKT- and ERK-dependent activation of NF-κB pathway. Oncotarget. 2014;5:8778–89.
Cai S, Ye Z, Wang X, Pan Y, Weng Y, Lao S, et al. Overexpression of P21-activated kinase 4 is associated with poor prognosis in non-small cell lung cancer and promotes migration and invasion. J Exp Clin Cancer Res. 2015;34:48.
Shu X-R, Wu J, Sun H, Chi L-Q, Wang J-H. PAK4 confers the malignance of cervical cancers and contributes to the cisplatin-resistance in cervical cancer cells via PI3K/AKT pathway. Diagn Pathol. 2015;10:177.
Bi Y, Tian M, Le J, Wang L, Liu X, Qu J, et al. Study on the expression of PAK4 and P54 protein in breast cancer. World J Surg Oncol. 2016;14:160.
Thillai K, Sarker D, Wells C. PAK4 pathway as a potential therapeutic target in pancreatic cancer. Future Oncol. 2018;14:579–82.
Wang M, Gao Q, Chen Y, Li Z, Yue L, Cao Y. PAK4, a target of miR-9-5p, promotes cell proliferation and inhibits apoptosis in colorectal cancer. Cell Mol Biol Lett. 2019;24:58.
Kim SH, Kim SR, Ihm HJ, Oh YS, Chae HD, Kim CH, et al. Regulation of P21-activated kinase-4 by progesterone and tumor necrosis factor-α in human endometrium and its increased expression in advanced-stage endometriosis. J Clin Endocrinol Metab. 2013;98:E238–48.
Murray BW, Guo C, Piraino J, Westwick JK, Ahzng C, Lamerdin J, et al. Small-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growth. Proc Natl Acad Sci U S A. 2010;107:9446–51.
Zhang J, Wang J, Guo Q, Wang Y, Zhou Y, Peng H, et al. LCH-7749944, a novel and potent p21-activated kinase 4 inhibitor, suppresses proliferation and invasion in human gastric cancer cells. Cancer Lett. 2012;317:24–32.
Hao C, Zhao F, Song H, Guo J, Li X, Jiang X, et al. Structure-based design of 6-chloro-4-aminoquinazoline-2-carboxamide derivatives as potent and selective p21-activated kinase 4 (PAK4) inhibitors. J Med Chem. 2018;61:265–85.
Li Y, Jia S, Dai W. Fisetin modulates human oral squamous cell carcinoma proliferation by blocking PAK4 signaling pathways. Drug Design Dev Ther. 2020;14:773–82.
Zhang H-Y, Zhang J, Hao C-Z, Zhou Y, Wang J, Cheng M-S, et al. LC-0882 targets PAK4 and inhibits PAK4-related signaling pathways to suppress the proliferation and invasion of gastric cancer cells. Am J Transl Res. 2017;9:2736–47.
Liu X, Duan H, Liu Z, Wu D, Zhao T, Zu S, et al. microRNA-199a-3p functions as tumor suppressor by regulating glucose metabolism in testicular germ cell tumors. Mol Med Rep. 2016;14:2311–20.
Wang X, Ren R, Shao M, Lan J. MicroRNA-16 inhibits endometrial stromal cell migration and invasion through suppression of the inhibitor of nuclear factor-κB kinase subunit β/nuclear factor-κB pathway. Int J Mol Med. 2020;46:740–50.
Shen L, Yang S, Huang W, Xu W, Wang Q, Song Y, et al. MicroRNA23a and microRNA23b deregulation derepresses SF-1 and upregulates estrogen signaling in ovarian endometriosis. J Clin Endocrinol Metab. 2013;98:1575–82.
Long M, Wan X, La X, Gong X, Cai X. miR-29c is downregulated in the ectopic endometrium and exerts its effects on endometrial cell proliferation, apoptosis and invasion by targeting c-Jun. Int J Mol Med. 2015;35:1119–25.
Yang WW, Hong L, Xu XX, Wang Q, Huang JL, Jiang L. Regulation of miR-33b on endometriosis and expression of related factors. Eur Rev Med Pharmacol Sci. 2017;21:2027–33.
Luo Y, Wang D, Chen S, Yang Q. The role of miR-34c-5p/Notch in epithelial-mesenchymal transition (EMT) in endometriosis. Cell Signal. 2020;72:109666.
Lv X, Chen P, Liu W. Down regulation of MiR-93 contributes to endometriosis through targeting MMP3 and VEGFA. Am J Cancer Res. 2015;5:1706–17.
Meng X, Liu J, Wang H, Chen P, Wang D. MicroRNA-126-5p downregulates BCAR3 expression to promote cell migration and invasion in endometriosis. Mol Cell Endocrinol. 2019;494:110486.
Zhang Y, Yan J, Pan X. miR-141-3p affects apoptosis and migration of endometrial stromal cells by targeting KLF-12. Pflugers Arch. 2019;471:1055–63.
Ma L, Li Z, Li W, Ai J, Chen X. MicroRNA-142-3p suppresses endometriosis by regulating KLF9-mediated autophagy in vitro and in vivo. RNA Biol. 2019;16:1733–48.
Adammek M, Greve B, Kässens N, Schneider C, Brüggemann K, Schüring AN, et al. MicroRNA miR-145 inhibits proliferation, invasiveness, and stem cell phenotype of an in vitro endometriosis model by targeting multiple cytoskeletal elements and pluripotency factors. Fertil Steril. 2013;99:1346–55.e5.
Shi X-Y, Gu L, Chen J, Guo X-R, Shi Y-L. Downregulation of miR-183 inhibits apoptosis and enhances the invasive potential of endometrial stromal cells in endometriosis. Int J Mol Med. 2014;33:59–67.
Hsu CY, Hsieh TH, Tsai CF, Tsai HP, Chen HS, Chang Y, et al. miRNA-199a-5p regulates VEGFA in endometrial mesenchymal stem cells and contributes to the pathogenesis of endometriosis. J Pathol. 2014;232:330–43.
Eggers JC, Martino V, Reinbold R, Schäfer SD, Kiesel L, Starzinski-Powitz A, et al. microRNA miR-200b affects proliferation, invasiveness and stemness of endometriotic cells by targeting ZEB1, ZEB2 and KLF4. Reprod BioMed Online. 2016;32:434–45.
Liang Z, Chen Y, Zhao Y, Xu C, Zhang A, Zhang Q, et al. miR-200c suppresses endometriosis by targeting MALAT1 in vitro and in vivo. Stem Cell Res Ther. 2017;8:251.
Zhang D, Li Y, Tian J, Zhang H, Wang S. MiR-202 promotes endometriosis by regulating SOX6 expression. Int J Clin Exp Med. 2015;8:17757–64.
Zhang M, Zhang Y, Li L, Ma L, Zhou C. Dysregulation of miR-202-3p affects migration and invasion of endometrial stromal cells in endometriosis via targeting ROCK1. Reprod Sci. 2020;27:731–42.
Zhou C-F, Liu M-J, Wang W, Wu S, Huang Y-X, Chen G-B, et al. miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells. Stem Cell Res Ther. 2019;10:287.
Wu D, Lu P, Mi X, Miao J. Exosomal miR-214 from endometrial stromal cells inhibits endometriosis fibrosis. Mol Hum Reprod. 2018;24:357–65.
Liu Y, Chen J, Zhu X, Tang L, Luo X, Shi Y. Role of miR-449b-3p in endometriosis via effects on endometrial stromal cell proliferation and angiogenesis. Mol Med Rep. 2018;18:3359–65.
Yu H, Zhong Q, Xia Y, Li E, Wang S, Ren R. MicroRNA-2861 targets STAT3 and MMP2 to regulate the proliferation and apoptosis of ectopic endometrial cells in endometriosis. Pharmazie. 2019;74:243–9.
Acknowledgements
We would like to thank Ms. Sawako Adachi, Ms. Tomoko Ohkuma, and Ms. Nozomi Kai for their excellent technical assistance and Editage (www.editage.jp) for English language editing.
Funding
This work was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 20K09622 and no. 16K11093 to K.N., no. 18K16774 to T.H., and no. 15K10679 to H.N.) and the Study Fund of Oita Society of Obstetrics and Gynecology (to T.H. and Y.A.).
Author information
Authors and Affiliations
Contributions
R.Z. and K.N. participated in the study design, data analysis and interpretation, literature search, generation of figures, and writing and editing of the manuscript. N.H., M.Y., T.H., Y.A., and H.N. performed the data/case collection, experiments, data analysis, and interpretation. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Ethical Approval
The study was approved by the Institutional Review Board of the Faculty of Medicine, Oita University (registration number: P-16-01).
Consent to Participate
All subjects provided informed written consent.
Conflict of Interest
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.
Rights and permissions
About this article
Cite this article
Zhu, R., Nasu, K., Hijiya, N. et al. hsa-miR-199a-3p Inhibits Motility, Invasiveness, and Contractility of Ovarian Endometriotic Stromal Cells. Reprod. Sci. 28, 3498–3507 (2021). https://doi.org/10.1007/s43032-021-00604-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s43032-021-00604-4