{"paper_id":"c705b12f-b3af-4c5b-a1ed-26c190f0aae5","body_text":"ORIGINAL ARTICLES\nPharmazie 74 (2019) 243\nDepartment of Gynecology1, Lishui Maternal and Child Health Care Center, Lishui, Zhejiang; Department of Obstetrics \nDelivery Room2, Zhangqin People’s Hospital, Jinan, Shandong; Department of Obstetrics and Gynecology3, Xianyang \nHospital, Y an’an University, Xianyang, Shaanxi; Department of Oncology4, Obstetrics and Gynecology Hospital, Zhejiang \nUniversity School of Medicine, Zhejiang; Department of Gynecology5, Xi’an Ninth Hospital, Xi’an, Shaanxi, China\nMicroRNA-2861 targets STAT3 and MMP2 to regulate the proliferation and \napoptosis of ectopic endometrial cells in endometriosis\nHAO YU1, QUN ZHONG2, YALI XIA3, ENCHUN LI4, SHUFANG WANG1, RONG REN5,*\nReceived November 10, 2018, accepted December 22, 2018\n*Corresponding author: Rong Ren, Department of Gynecology, Xi’an Ninth Hospital, No.151 East Section of \nNan’erhuan Road, Xi’an, Shaanxi 710054, China.\nrongren02@126.com\nPharmazie 74: 243-249 (2019) doi: 10.1691/ph.2019.8881\nThis study aimed to elucidate the roles and regulatory mechanism of miR-2861 in the development of endometri-\nosis. The expression of miR-2861 in endometriotic tissues and eutopic endometrial tissues was determined. Ectopic \nendometrial cells were transfected with miR-2861 mimic, miR-2861 inhibitor and their corresponding controls. The \neffects of miR-2861 overexpression and inhibition on cell proliferation and apoptosis were then investigated. In \naddition, regulatory relationship between miR-2681 and signal transducer and activator of transcription 3 (STAT3) \nor matrix metalloproteinase 2 (MMP2) was explored, as well as between STAT3 and MMP2. Compared with \neutopic endometrial tissues, miR-2861 was significantly downregulated in endometriotic tissues. Overexpression \nof miR-2861 markedly inhibited ectopic endometrial cell proliferation and induced apoptosis. Additionally, STAT3 \nand MMP2 were confirmed as the targets of miR-2861, and the effects of knockdown of STAT3 or MMP2on regu-\nlating cell proliferation and apoptosis were opposite with inhibition of miR-2861 . Besides, STAT3 could enhance \nthe expression of MMP2 in ectopic endometrial cells. Our study reveals that miR-2861 was lowly expressed in \nendometriotic tissues, and downregulation of miR-2861 may promote proliferation and inhibit apoptosis of ectopic \nendometrial cells in endometriosis via upregulation of STAT3 and MMP2. miR-2861 may be a potential biomarker \nor therapeutic target for endometriosis. \n1. Introduction\nEndometriosis is an estrogen-dependent inflammatory disease, \nwhich is characterized by endometrial-type mucosa outside the \nuterine cavity (Iwabe and Harada 2014; Vercellini et al. 2014). \nThe prevalence of endometriotic disease is approximately 5% and \nreaches it’s peak between the ages of 25 and 35 years (Giudice \net al. 2012; Viganò et al. 2004). Endometriosis is always accom-\npanied by several pain symptoms, including dysmenorrhoea, low \nback pain, dyspareunia, dysuria and dyschezia (Tokushige 2014). \nCurrent treatments of endometriosis are surgery and ovarian \nsuppressive agents, including GnRh agonists, progestins, oral \ncontraceptives, and androgenic agents; however, hormonal treat-\nments are associated with side effects, such as delayed conception \n(Elnashar 2015). Seriously, endometriosis is found responsible for \na 50 % increase in the risk of epithelial ovarian cancer (Vercellini \net al. 2014). Therefore, it is still necessary to look for effective \ntreatments for endometriosis.\nMicroRNAs (miRNAs) are small, non-coding RNAs with lengths \nof 20-25 nucleotides and have been reported to be implicated in \nmultiple physiologic and pathologic processes via modulating \nthe gene expression of specific targets (Kloosterman and Plas-\nterk 2006; O’Connell et al. 2010). Dysregulation of  miRNAs is \nproposed to be key regulators in various cellular processes that \noccur in endometriosis (Braza-Boïls et al. 2013, 2014; Laudanski \net al. 2013; Nothnick 2017). In addition, miRNAs have emerged \nas potential biomarkers for the diagnosis of endometriosis (Cho et \nal. 2015; Cosar et al. 2016). Recently, miR-2861 has been found \nto play a role in regulating the development of papillary thyroid \ncarcinoma (Wang et al. 2013). It is reported that miR-2861 plays \na tumor suppressor role in human papillomavirus virus 16 E6-in-\nduced cervical cancer via targeting the EGFR/AKT2/CCND1 \npathway (Xu et al. 2016). miR-2861 is also identified as a non-in-\nvasive biomarker for detecting cervical cancer (Zhang et al. 2015). \nDespite these, there were limited reports on the roles of miR-2861 \nin endometriosis.\nIn this study, we analyzed the expression of miR-2861 in endome-\ntriotic tissues and investigated the effects of miR-2861 dysregula-\ntion on cell proliferation and apoptosis. In addition, the regulatory \nrelationship between miR-2681 and signal transducer and activator \nof transcription 3 (STAT3) or matrix metalloproteinase 2 (MMP2) \nwas explored to further elucidate the mechanism of miR-2681 on \nendometriosis. Our findings will provide a theoretical basis for a \nbetter understanding of the molecular mechanism of endometri-\nosis.\n2. Investigations and results\n2.1. miR-2861 was downregulated in endometriotic \ntissues\nTo investigate whether miR-2861 was a key regulator in the \ndevelopment of endometriosis, the expression of miR-2861 was \nmeasured by qRT-PCR. The results show that miR-2861 was \ndownregulated in endometriotic tissues compared with eutopic \nendometrial tissues (P<0.05, Fig. 1A), indicating that miR-2861 \nmay play a key role in endometriosis development.\n2.2. miR-2861 was successfully overexpressed and \nsuppressed in ectopic endometrial cells\nTo explore the role of miR-2861 in endometriosis development, \nectopic endometrial cells were transfected with miR-2861 mimic, \nmiR-2861 inhibitor and their corresponding controls. In comparison \nwith their corresponding controls, cell viability of miR-2861 mimic \ntransfected cells was significantly decreased with the increase of \n\nORIGINAL ARTICLES\nPharmazie 74 (2019)244\ntransfection time (P<0.05, Fig. 1B), while cell viability of miR-2861 \ninhibitor transfected cells was gradually promoted (P<0.05, Fig. \n1C). Moreover, the expression of miR-2861 was markedly increased \nafter transfection with miR-2861 mimic, while obvious decreased \nafter transfection with miR-2861 inhibitor (P<0.05, Fig. 1D). These \ndata confirmed that miR-2861 was successfully overexpressed and \nsuppressed in ectopic endometrial cells.\n2.3. Inhibition of miR-2861 promoted proliferation and \ninhibited apoptosis of ectopic endometrial cells\nAs shown in Figs. 2A and 2B, the BrdU-positive cells in miR-2861 \nmimic group were significantly decreased compared with the \nmimic control group, while the BrdU-positive cells in miR-2861 \ninhibitor group were markedly increased compared with inhibitor \ncontrol group (P<0.05). Moreover, in comparison with their corre-\nsponding control groups, the apoptotic cells were significantly \nincreased in miR-2861 mimic group, while markedly decreased in \nmiR-2861 inhibitor group (P<0.05, Figure 2C and 2D). Besides, \nthe expression ratio of BAX/BCL2 was enhanced after transfection \nwith miR-2861 mimic, while obviously decreased after transfec-\ntion with miR-2861 inhibitor (P<0.05, Figs. 2E and 2F).\n2.4. STAT3 and MMP2 were directly targets of miR-2861\nThe potential targets of miR-2861 were also predicted using the \nonline database TargetScanHuman 7.1 (http://www.targetscan.org/\nvert_71/), including STAT3 and MMP2 (Fig. 3A). To confirm the \npredicted results, luciferase reporter assay was carried out and the \nresults showed that miR-2861 mimic significantly inhibited the \nluciferase activity of STAT3 or MMP2 3’UTR-wt (P < 0.05, Figs. \n3B and 3C), but could not target STAT3 or MMP2 3’UTR-mut. \nCompared with their corresponding control groups, the expres-\nsion levels of STAT3 and MMP2 were significantly decreased in \nmiR-2861 mimic group, while markedly increased in miR-2861 \ninhibitor group (P < 0.05, Figs. 3D and 3E). Besides, the expres-\nsion levels of STAT3 and MMP2 in endometriotic tissues were \nsignificantly higher than that in eutopic endometrial tissues (P < \n0.05, Figs. 3F and 3G). These findings confirmed that STAT3 and \nMMP2 were directly targets of miR-2861.\n2.5. Knockdown of STAT3 and MMP2 inhibited prolifer-\nation and induced apoptosis of ectopic endometrial cells\nTo further confirm whether STAT3 and MMP2 were involved in \nthe regulatory roles of miR-2861 in ectopic endometrial cells, the \nexpression levels of STAT3 and MMP2 were knocked down by \nsi-STAT3 and si-MMP2 (P < 0.05, Fig. 4A), respectively. More-\nover, the BrdU-positive cells in si-STAT3 and si-MMP2 groups \nwere all significantly decreased compared with scramble control \ngroup (P<0.05, Fig. 4B). However, the apoptotic cells in si-STAT3 \nand si-MMP2 groups were significantly increased compared with \nscramble control group (P<0.05, Fig. 4C), as well as expression \nratio of BAX/BCL2 (P<0.05, Fig. 4D).\n2.6. STAT3 enhanced the expression of MMP2 in ectopic \nendometrial cells\nMMP2 expression was also significantly downregulated after \nknockdown of STAT3 (P < 0.05, Fig. 4A), implying that STAT3 \nmight inhibit the expression of MMP2. Additionally, the effects of \nSTAT3 knockdown on the proliferation and apoptosis of ectopic \nendometrial cells were more obvious than the effects of MMP2 \nknockdown (P < 0.05, Fig. 4A-D). To verify the enhanced effects \nFig. 1: miR-2861 was downregulated in endometriotic tissues and miR-2861 was successfully overexpressed and suppressed in ectopic endometrial cells. A: The expression level of \nmiR-2861 in endometriotic tissues and eutopic endometrial tissues. B: MTT showed the cell viability of miR-2861 mimic and mimic control transfected cells. C: MTT showed \nthe cell viability of miR-2861 inhibitor and inhibitor control transfected cells. D: The expression of miR-2861 in different transfected groups. *p < 0.05; **p < 0.01. \n\nORIGINAL ARTICLES\nPharmazie 74 (2019) 245\nFig. 2: Inhibition of miR-2861 promoted proliferation and inhibited apoptosis of ectopic endometrial cells. A and B: The BrdU-p ositive cells in different transfected groups. \nC and D: Flow cytometry showed the apoptotic cells in different transfected groups. E and F: Western blot showed the expression  levels of BAX and BCL2 in different \ntransfected groups. *p < 0.05; **p < 0.01, *** p < 0.001.\nof STAT3 on MMP2 expression, ectopic endometrial cells were \ntransfected with pcDNA-STAT3, pcDNA-MMP2 and blank vector. \nThe results show that STAT3 and MMP2 were all upregulated in \nectopic endometrial cells after transfection with pcDNA-STAT3, \nwhile only MMP2 was upregulated in ectopic endometrial cells \nafter transfection with pcDNA-MMP2 (P < 0.05, Figs. 5A and 5B). \nThese data imply that STAT3 may promote MMP2 expression in \nectopic endometrial cells.\n3. Discussion\nEndometriosis is a common chronic disease in gynecology, yet its \npathogenesis remains largely unknown (Shi et al. 2014). In this \nstudy, we found that miR-2861 was significantly downregulated in \nendometriotic tissues compared with eutopic endometrial tissues. \nOverexpression of miR-2861 markedly inhibited ectopic endome-\ntrial cell proliferation and induced apoptosis, imply the inhibitory \neffects on endometriosis. Additionally, STAT3 and MMP2 were \nconfirmed as the targets of miR-2861, and knockdown of STAT3 \nor MMP2 had opposite effects with inhibition of miR-2861 on \nregulating cell proliferation and apoptosis. Besides, STAT3 may \npromote the expression of MMP2 in ectopic endometrial cells, \nthus to affect the effects of MMP2 on endometriosis. These find-\nings highlight the biological actions of miR-2861 in endometriosis \nand merit further discussion.\nmiRNAs play a vital role in the regulation of the endometriosis \ndevelopment, including miR-199a-5p (Hsu et al. 2014) and \nmiR-451(Graham et al. 2015). This study found the downregula-\ntion of miR-2861 in endometriotic tissues, however, few studies \nhave investigated the roles of miR-2861 in endometriosis. It has \nbeen reported that miR-2861 can inhibit cell proliferation and \npromote apoptosis in cervical cancer (Xu et al. 2016), indicating \nthat miR-2861 may act as a tumor suppressor in cervical cancer. \nSimilarly, we found that miR-2861 markedly inhibited ectopic \nendometrial cell proliferation and induced apoptosis, imply the \ninhibitory effects on endometriosis.\n\nORIGINAL ARTICLES\nPharmazie 74 (2019)246\nFig. 3: STAT3 and MMP2 were directly targets of miR-2861. A: The predicted target sequences of miR-2861 and STAT3 or MMP2 using the online database TargetScanHuman \n7.1. B and C: Luciferase reporter assay showed the luciferase activity of STAT3 or MMP2 3’UTR-wt and STAT3 or MMP2 3’UTR-mut after cotransfection with miR-\n2861 mimic or mimic control. D and E: Western blot showed the expression levels of STAT3 and MMP2 in different transfected groups. F and G: Western blot showed the \nexpression levels of STAT3 and MMP2 in endometriotic tissues and eutopic endometrial tissues. **p < 0.01, *** p < 0.001.\nFurthermore, this study confirmed the target genes of miR-2861 \nwere STAT3 and MMP2. In women with endometriosis, STAT3 \nactivation may be central to the inflammatory phenotype of eutopic \nendometrium (Yoo et al. 2016). It has also been reported that aber-\nrant activation of STAT3 signaling plays a key role in the pathogen-\nesis of endometriosis (Kim et al. 2015). Moreover, the interaction \nbetween endometrial stromal cells and peritoneal M2 macrophages \ncontributes to endometriosis development by activation of STAT3 \n(Itoh et al. 2014). Oncostatin M is found to promote tumor invasion \nand angiogenesis in endometrial cancer through activating of STAT3 \n(Zhu et al. 2015). In this study, knockdown of STAT3 inhibited \nproliferation and induced apoptosis of ectopic endometrial cells, \nwhich was opposite with the effects of miR-2861 inhibition. There-\nfore, we speculate that downregulation of miR-2861 may promote \nendometriosis development via activation of STAT3. \nOn the other hand, MMP2 is found to be related with the changes \nin steroid hormones, thus resulting in the formation of endome-\ntriosis (Huang et al. 2004). Malvezzi et al. (2013) also demon-\nstrated that the severity of advanced pelvic endometriosis was \nrelated to higher serum MMP-2 levels (Malvezzi et al. 2013). \nA miR-520 mirSNP at the MMP2 gene is confirmed to affect \nthe susceptibility to endometriosis in Chinese women (Tsai et al. \n\nORIGINAL ARTICLES\nPharmazie 74 (2019) 247\n2013). In our study, the effects of knockdown of STAT3 on prolif-\neration and apoptosis of ectopic endometrial were also opposite \nwith the effects of miR-2861 inhibition. These data indicate \nthat downregulation of miR-2861 may promote endometriosis \ndevelopment via regulating MMP2. Notably, our results also \nshowed that MMP2 expression was significantly downregulated \nafter knockdown of STAT3, and STAT3 and MMP2 were all \nupregulated in ectopic endometrial cells after transfection with \npcDNA-STAT3. The effects of STAT3 knockdown on the prolif-\neration and apoptosis of ectopic endometrial cells were more \nobvious than the effects of MMP2 knockdown. Leptin is reported \nto promote the migration and invasion of endometriotic cells by \nupregulation of MMP-2 via the JAK2/STAT3 signaling pathway \n(Ahn et al. 2015). Taken together, we hypothesize that miR-2861 \nmay promote endometriosis development via regulating MMP2 \nthrough activation of STAT3 signaling.\nIn conclusion, our study reveals that miR-2861 is lowly expressed \nin endometriotic tissues and downregulation of miR-2861 may \npromote endometriosis development via upregulation of STAT3 and \nMMP2. These findings suggest that miR-2861 may be a potential \nbiomarker or therapeutic target for endometriosis. Further experi-\nmental studies are still needed for the verification of our findings.\n4. Experimental\n4.1. Tissue sampling\nUnder a protocol approved by local committee, this study was conducted. All participants \nprovided written informed consent for participation. A total of 19 patients who suffered \nfrom endometriosis (mean age 39.3 years; range 34-43 years) were enrolled into this study, \nand their eutopic endometrial and endometriotic tissues were obtained by laparoscopy. \nSix months before the surgical operation, patients had not received any hormonal drugs, \nsuch as GnRH analog or other. All the samples were confirmed histologically. Eutopic \nendometrium acquired from 12 disease-free women was considered as healthy controls.\nFig. 4: Knockdown of STAT3 and MMP2 inhibited proliferation and induced apoptosis of ectopic endometrial cells. A: The expression levels of STAT3 and MMP2 after transfec-\ntion with si-STAT3, si-MMP2 and scramble control. B: The BrdU-positive cells in different transfected groups. C: Flow cytometry showed the apoptotic cells in different \ntransfected groups. D: Western blot showed the expression levels of BAX and BCL2 in different transfected groups. *p < 0.05; **p < 0.01, *** p < 0.001.\n\nORIGINAL ARTICLES\nPharmazie 74 (2019)248\nFig. 5: The expression levels of STAT3 and MMP2 in ectopic endometrial cells \nwhich were transfected with pcDNA-STAT3, pcDNA-MMP2 and blank vec-\ntor. **p < 0.01, *** p < 0.001.\n4.2. Cell isolation and culture\nThe endometriotic tissues were cut to little pieces and then incubated with enzymatic \ndissociation solution for 10 min. After discarding the dissociation solution, the \nisolated ectopic endometrial cells were resuspended with compete Dulbecco’s Modi-\nfied Eagle Medium (DMEM) containing 10 % fetal calf serum in a 95 % humidified \nincubator 37 °C, 5 % CO\n2.\n4.3. Cell transfection\nThe cells were transfected with miR-29c mimic, miR-29c inhibitor, and their corre-\nsponding controls, mutant and wild-type STAT3 or MMP2 3’UTR, siRNA for STAT3 \nor MMP2, and the overexpression plasmid of STAT3 or MMP2 using Lipofectamine \n2000 reagent (Invitrogen) according to the manufacturer’s instructions and then incu-\nbated at 37 °C for 24 h.\n4.4. MTT assay\nCell viability was evaluated with a MTT assay. The cells were seeded into 96-well \nculture plates at a density of 1x10\n4 cells/well. At 12, 24 and 48 after transfection, to \neach well 10 μl MTT solution (0.5 mg/ml; Beyotime, Shanghai, China) was added and \nincubation was continued for 4 h. Afterwards, DMSO (Sigma, St. Louis, MO, USA) \nwas added to dissolve the MTT formazan. Cell viability of each well was read at \n570 nm on a Multiskan Ascent 354 microplate reader (Thermo Labsystems, Waltham, \nMA, USA).\n4.5. BrdU cell proliferation assay\nCells were harvested, washed with PBS, fixed with 4 % paraformaldehyde for 15 \nmin, permeabilized with 0.1 % Triton/PBS, denatured with 0.1 N HCl, washed in \nPBST (1# PBS Tween-20), blocked with 5 % BSA/PBST, and then incubated with \nthe anti-BrdU antibody (1:100, Sigma, USA). After washing with PBST for three \ntimes, cells were incubated with the appropriate Cy-2- or Cy-3–conjugated secondary \nantibody (Jackson Immunoresearch) away from the light for 45 min. Cells were \ncounterstained with DAPI, mounted with antifade (Vectastain), and visualized by \nfluorescence microscopy.\n4.6. Flow cytometry analysis\nCell apoptosis was assessed by flow cytometry. Briefly, cells were resuspended in \nbinding buffer and then double stained with Annexin V and propidium iodide (PI) \n(Kaiji Biological Inc., Nanjing, China) according to the manufacturer’s instructions \nof a FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Shanghai, China). \nApoptotic cells were then detected and analyzed by flow cytometer (BD FACSAria; \nBD Biosciences, Franklin Lakes, NJ, USA).\n4.7. Luciferase report assay\nThe 3’-UTRs of human STAT3 and MMP2 and their corresponding mutated 3’-UTRs \nwere amplified by PCR. Cells (2×10 4) were seeded into a 48-well plate. Sequences \nfrom the miR-2861 target site in the 3’-UTR of STAT3 and MMP2 and their mutant \nvariants were cloned into the psiCHECK-2 vector (Promega), which were then \ncontransfected with miR-2861 mimic or mimic control into cells. 48 h after trans-\nfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay \nSystem (Promega). Renilla luciferase activity was used as the internal control.\n4.8. Quantitative real-time PCR (qRT-PCR)\nFollowing the manufacturer’s instructions, the miRNAs was extracted by a miRNeasy \nMini kit (Qiagen, Hilden, Germany). Reverse transcription into cDNA was then \nconducted with a RevertAid™ First Strand cDNA Synthesis kit (Fermentas, Vilnius, \nLithuania). To detect the expression of miR-2861, qRT-PCR was performed using \na SYBRGreen PCR kit (both from Applied Biosystems, Foster City, CA, USA) by \nmeans of a 7900 Real-Time PCR System. After normalization with the expression of \nU6, the relative expression of miR-2861was analyzed using the comparative (2\n-ΔΔCt ) \nmethod.\n4.9. Western blot\nTotal protein was extracted from cells using a total protein extraction kit (Kaiji \nBiological, Inc.), which concentration was then measured with the BCA kit (Beyo-\ntime). The equal amount of samples was then separated in 10 % SDS-PAGE and \nblotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). \nAfter blocked in 5 % non-fat milk, the membranes were incubated with primary anti-\nbodies, including mouse monoclonal to STAT3, MMP2 and GAPDH (1:1000, Abcam, \nCambridge, MA, USA) overnight at 4 °C, followed by incubation with secondary \nantibody conjugated with horseradish peroxidase (1:5000; BA1050; Boster, Wuhan, \nHubei, China) for 2 h. GAPDH was used as a control. The ECL detection kit (Pierce, \nRockford, IL, USA) was used to visualize the protein blots, and the densitometry of \nproteins was then analyzed with Quantity One software (Bio-Rad, CA, USA). \n4.10. Statistical analysis\nAll experiments were performed three times at least, and the related data were shown \nas the mean±SD. Data were analyzed with Student’s t-test or one-way analysis of vari-\nance (ANOV A) in Statistical Package for the Social Sciences (SPSS) software version \n11.5 (IBM, IL, USA). Statistically significant differences were obtained at P<0.05.\nConflicts of interest: None reported.\nReferences\nAhn J-H, Choi YS, Choi JH (2015) Leptin promotes human endometriotic cell migra-\ntion and invasion by upregulating MMP-2 through the JAK2/STAT3 signaling \npathway. MHR: Basic Sci Reprod Med 21: 792-802.\nBraza-Boïls A, Gilabert-Estellés J, Ramón LA, Gilabert J, Marí-Alexandre J, Chiriv-\nella M, España F, Estellés A (2013) Peritoneal fluid reduces angiogenesis-related \nmicroRNA expression in cell cultures of endometrial and endometriotic tissues \nfrom women with endometriosis. 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