MicroRNA-2861 targets STAT3 and MMP2 to regulate the proliferation and apoptosis of ectopic endometrial cells in endometriosis

showed that miR-2861 mimic significantly inhibited the luciferase activity of STAT3 or MMP2 3’UTR-wt (P < 0.05, Figs. 3B and 3C), but could not target STAT3 or MMP2 3’UTR-mut. Compared with their corresponding control groups, the expres- sion levels of STAT3 and MMP2 were significantly decreased in miR-2861 mimic group, while markedly increased in miR-2861 inhibitor group (P < 0.05, Figs. 3D and 3E). Besides, the expres- sion levels of STAT3 and MMP2 in endometriotic tissues were significantly higher than that in eutopic endometrial tissues (P < 0.05, Figs. 3F and 3G). These findings confirmed that STAT3 and MMP2 were directly targets of miR-2861. 2.5. Knockdown of STAT3 and MMP2 inhibited prolifer- ation and induced apoptosis of ectopic endometrial cells To further confirm whether STAT3 and MMP2 were involved in the regulatory roles of miR-2861 in ectopic endometrial cells, the expression levels of STAT3 and MMP2 were knocked down by si-STAT3 and si-MMP2 (P < 0.05, Fig. 4A), respectively. More- over, the BrdU-positive cells in si-STAT3 and si-MMP2 groups were all significantly decreased compared with scramble control group (P<0.05, Fig. 4B). However, the apoptotic cells in si-STAT3 and si-MMP2 groups were significantly increased compared with scramble control group (P<0.05, Fig. 4C), as well as expression ratio of BAX/BCL2 (P<0.05, Fig. 4D). 2.6. STAT3 enhanced the expression of MMP2 in ectopic endometrial cells MMP2 expression was also significantly downregulated after knockdown of STAT3 (P < 0.05, Fig. 4A), implying that STAT3 might inhibit the expression of MMP2. Additionally, the effects of STAT3 knockdown on the proliferation and apoptosis of ectopic endometrial cells were more obvious than the effects of MMP2 knockdown (P < 0.05, Fig. 4A-D). To verify the enhanced effects Fig. 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 miR-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 the 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. ORIGINAL ARTICLES Pharmazie 74 (2019) 245 Fig. 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. C 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 transfected groups. *p < 0.05; **p < 0.01, *** p < 0.001. of STAT3 on MMP2 expression, ectopic endometrial cells were transfected with pcDNA-STAT3, pcDNA-MMP2 and blank vector. The results show that STAT3 and MMP2 were all upregulated in ectopic endometrial cells after transfection with pcDNA-STAT3, while only MMP2 was upregulated in ectopic endometrial cells after transfection with pcDNA-MMP2 (P < 0.05, Figs. 5A and 5B). These data imply that STAT3 may promote MMP2 expression in ectopic endometrial cells. 3. Discussion Endometriosis is a common chronic disease in gynecology, yet its pathogenesis remains largely unknown (Shi et al. 2014). In this study, we found that miR-2861 was significantly downregulated in endometriotic tissues compared with eutopic endometrial tissues. Overexpression of miR-2861 markedly inhibited ectopic endome- trial cell proliferation and induced apoptosis, imply the inhibitory effects on endometriosis. Additionally, STAT3 and MMP2 were confirmed as the targets of miR-2861, and knockdown of STAT3 or MMP2 had opposite effects with inhibition of miR-2861 on regulating cell proliferation and apoptosis. Besides, STAT3 may promote the expression of MMP2 in ectopic endometrial cells, thus to affect the effects of MMP2 on endometriosis. These find- ings highlight the biological actions of miR-2861 in endometriosis and merit further discussion. miRNAs play a vital role in the regulation of the endometriosis development, including miR-199a-5p (Hsu et al. 2014) and miR-451(Graham et al. 2015). This study found the downregula- tion of miR-2861 in endometriotic tissues, however, few studies have investigated the roles of miR-2861 in endometriosis. It has been reported that miR-2861 can inhibit cell proliferation and promote apoptosis in cervical cancer (Xu et al. 2016), indicating that miR-2861 may act as a tumor suppressor in cervical cancer. Similarly, we found that miR-2861 markedly inhibited ectopic endometrial cell proliferation and induced apoptosis, imply the inhibitory effects on endometriosis. ORIGINAL ARTICLES Pharmazie 74 (2019)246 Fig. 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 7.1. 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- 2861 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 expression levels of STAT3 and MMP2 in endometriotic tissues and eutopic endometrial tissues. **p < 0.01, *** p < 0.001. Furthermore, this study confirmed the target genes of miR-2861 were STAT3 and MMP2. In women with endometriosis, STAT3 activation may be central to the inflammatory phenotype of eutopic endometrium (Yoo et al. 2016). It has also been reported that aber- rant activation of STAT3 signaling plays a key role in the pathogen- esis of endometriosis (Kim et al. 2015). Moreover, the interaction between endometrial stromal cells and peritoneal M2 macrophages contributes to endometriosis development by activation of STAT3 (Itoh et al. 2014). Oncostatin M is found to promote tumor invasion and angiogenesis in endometrial cancer through activating of STAT3 (Zhu et al. 2015). In this study, knockdown of STAT3 inhibited proliferation and induced apoptosis of ectopic endometrial cells, which was opposite with the effects of miR-2861 inhibition. There- fore, we speculate that downregulation of miR-2861 may promote endometriosis development via activation of STAT3. On the other hand, MMP2 is found to be related with the changes in steroid hormones, thus resulting in the formation of endome- triosis (Huang et al. 2004). Malvezzi et al. (2013) also demon- strated that the severity of advanced pelvic endometriosis was related to higher serum MMP-2 levels (Malvezzi et al. 2013). A miR-520 mirSNP at the MMP2 gene is confirmed to affect the susceptibility to endometriosis in Chinese women (Tsai et al. ORIGINAL ARTICLES Pharmazie 74 (2019) 247 2013). In our study, the effects of knockdown of STAT3 on prolif- eration and apoptosis of ectopic endometrial were also opposite with the effects of miR-2861 inhibition. These data indicate that downregulation of miR-2861 may promote endometriosis development via regulating MMP2. Notably, our results also showed that MMP2 expression was significantly downregulated after knockdown of STAT3, and STAT3 and MMP2 were all upregulated in ectopic endometrial cells after transfection with pcDNA-STAT3. The effects of STAT3 knockdown on the prolif- eration and apoptosis of ectopic endometrial cells were more obvious than the effects of MMP2 knockdown. Leptin is reported to promote the migration and invasion of endometriotic cells by upregulation of MMP-2 via the JAK2/STAT3 signaling pathway (Ahn et al. 2015). Taken together, we hypothesize that miR-2861 may promote endometriosis development via regulating MMP2 through activation of STAT3 signaling. In conclusion, our study reveals that miR-2861 is lowly expressed in endometriotic tissues and downregulation of miR-2861 may promote endometriosis development via upregulation of STAT3 and MMP2. These findings suggest that miR-2861 may be a potential biomarker or therapeutic target for endometriosis. Further experi- mental studies are still needed for the verification of our findings. 4. Experimental 4.1. Tissue sampling Under a protocol approved by local committee, this study was conducted. All participants provided written informed consent for participation. A total of 19 patients who suffered from endometriosis (mean age 39.3 years; range 34-43 years) were enrolled into this study, and their eutopic endometrial and endometriotic tissues were obtained by laparoscopy. Six months before the surgical operation, patients had not received any hormonal drugs, such as GnRH analog or other. All the samples were confirmed histologically. Eutopic endometrium acquired from 12 disease-free women was considered as healthy controls. Fig. 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- tion 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 transfected 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. ORIGINAL ARTICLES Pharmazie 74 (2019)248 Fig. 5: The expression levels of STAT3 and MMP2 in ectopic endometrial cells which were transfected with pcDNA-STAT3, pcDNA-MMP2 and blank vec- tor. **p < 0.01, *** p < 0.001. 4.2. Cell isolation and culture The endometriotic tissues were cut to little pieces and then incubated with enzymatic dissociation solution for 10 min. After discarding the dissociation solution, the isolated ectopic endometrial cells were resuspended with compete Dulbecco’s Modi- fied Eagle Medium (DMEM) containing 10 % fetal calf serum in a 95 % humidified incubator 37 °C, 5 % CO 2. 4.3. Cell transfection The cells were transfected with miR-29c mimic, miR-29c inhibitor, and their corre- sponding controls, mutant and wild-type STAT3 or MMP2 3’UTR, siRNA for STAT3 or MMP2, and the overexpression plasmid of STAT3 or MMP2 using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions and then incu- bated at 37 °C for 24 h. 4.4. MTT assay Cell viability was evaluated with a MTT assay. The cells were seeded into 96-well culture plates at a density of 1x10 4 cells/well. At 12, 24 and 48 after transfection, to each well 10 μl MTT solution (0.5 mg/ml; Beyotime, Shanghai, China) was added and incubation was continued for 4 h. Afterwards, DMSO (Sigma, St. Louis, MO, USA) was added to dissolve the MTT formazan. Cell viability of each well was read at 570 nm on a Multiskan Ascent 354 microplate reader (Thermo Labsystems, Waltham, MA, USA). 4.5. BrdU cell proliferation assay Cells were harvested, washed with PBS, fixed with 4 % paraformaldehyde for 15 min, permeabilized with 0.1 % Triton/PBS, denatured with 0.1 N HCl, washed in PBST (1# PBS Tween-20), blocked with 5 % BSA/PBST, and then incubated with the anti-BrdU antibody (1:100, Sigma, USA). After washing with PBST for three times, cells were incubated with the appropriate Cy-2- or Cy-3–conjugated secondary antibody (Jackson Immunoresearch) away from the light for 45 min. Cells were counterstained with DAPI, mounted with antifade (Vectastain), and visualized by fluorescence microscopy. 4.6. Flow cytometry analysis Cell apoptosis was assessed by flow cytometry. Briefly, cells were resuspended in binding buffer and then double stained with Annexin V and propidium iodide (PI) (Kaiji Biological Inc., Nanjing, China) according to the manufacturer’s instructions of a FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Shanghai, China). Apoptotic cells were then detected and analyzed by flow cytometer (BD FACSAria; BD Biosciences, Franklin Lakes, NJ, USA). 4.7. Luciferase report assay The 3’-UTRs of human STAT3 and MMP2 and their corresponding mutated 3’-UTRs were amplified by PCR. Cells (2×10 4) were seeded into a 48-well plate. Sequences from the miR-2861 target site in the 3’-UTR of STAT3 and MMP2 and their mutant variants were cloned into the psiCHECK-2 vector (Promega), which were then contransfected with miR-2861 mimic or mimic control into cells. 48 h after trans- fection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). Renilla luciferase activity was used as the internal control. 4.8. Quantitative real-time PCR (qRT-PCR) Following the manufacturer’s instructions, the miRNAs was extracted by a miRNeasy Mini kit (Qiagen, Hilden, Germany). Reverse transcription into cDNA was then conducted with a RevertAid™ First Strand cDNA Synthesis kit (Fermentas, Vilnius, Lithuania). To detect the expression of miR-2861, qRT-PCR was performed using a SYBRGreen PCR kit (both from Applied Biosystems, Foster City, CA, USA) by means of a 7900 Real-Time PCR System. After normalization with the expression of U6, the relative expression of miR-2861was analyzed using the comparative (2 -ΔΔCt ) method. 4.9. Western blot Total protein was extracted from cells using a total protein extraction kit (Kaiji Biological, Inc.), which concentration was then measured with the BCA kit (Beyo- time). The equal amount of samples was then separated in 10 % SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After blocked in 5 % non-fat milk, the membranes were incubated with primary anti- bodies, including mouse monoclonal to STAT3, MMP2 and GAPDH (1:1000, Abcam, Cambridge, MA, USA) overnight at 4 °C, followed by incubation with secondary antibody conjugated with horseradish peroxidase (1:5000; BA1050; Boster, Wuhan, Hubei, China) for 2 h. GAPDH was used as a control. The ECL detection kit (Pierce, Rockford, IL, USA) was used to visualize the protein blots, and the densitometry of proteins was then analyzed with Quantity One software (Bio-Rad, CA, USA). 4.10. Statistical analysis All experiments were performed three times at least, and the related data were shown as the mean±SD. Data were analyzed with Student’s t-test or one-way analysis of vari- ance (ANOV A) in Statistical Package for the Social Sciences (SPSS) software version 11.5 (IBM, IL, USA). Statistically significant differences were obtained at P<0.05. Conflicts of interest: None reported.

Ahn J-H, Choi YS, Choi JH (2015) Leptin promotes human endometriotic cell migra- tion and invasion by upregulating MMP-2 through the JAK2/STAT3 signaling pathway. MHR: Basic Sci Reprod Med 21: 792-802. Braza-Boïls A, Gilabert-Estellés J, Ramón LA, Gilabert J, Marí-Alexandre J, Chiriv- ella M, España F, Estellés A (2013) Peritoneal fluid reduces angiogenesis-related microRNA expression in cell cultures of endometrial and endometriotic tissues from women with endometriosis. Plos One 8: e62370. Braza-Boïls A, Marí-Alexandre J, Gilabert J, Sánchez-Izquierdo D, España F, Estellés A, Gilabert-Estellés J (2014) MicroRNA expression profile in endometriosis: its relation to angiogenesis and fibrinolytic factors. Human Reprod 29: 978-988. Cho S, Mutlu L, Grechukhina O, Taylor HS (2015) Circulating microRNAs as poten- tial biomarkers for endometriosis. Fertility Sterility 103: 1252-1260. Cosar E, Mamillapalli R, Ersoy GS, Cho S, Seifer B, Taylor HS (2016) Serum microRNAs as diagnostic markers of endometriosis: a comprehensive array-based analysis. Fertility Sterility 106: 402-409. Elnashar A (2015) Emerging treatment of endometriosis. Middle East Fertil Soc J 20: 61-69. Giudice LC, Evers JL, Healy DL (2012) Endometriosis: science and practice. John Wiley & Sons. Graham A, Falcone T, Nothnick WB (2015) The expression of microRNA-451 in human endometriotic lesions is inversely related to that of macrophage migration inhibitory factor (MIF) and regulates MIF expression and modulation of epithelial cell survival. Human Reprod 30: 642-652. Hsu CY , Hsieh TH, Tsai CF, Tsai HP, Chen HS, Chang Y , Chuang HY , Lee JN, Hsu YL, Tsai EM (2014) miRNA -199a-5p regulates VEGFA in endometrial mesen- chymal stem cells and contributes to the pathogenesis of endometriosis. J Pathol 232: 330-343. Huang H-F, Hong L-H, Tan Y , Sheng J-Z (2004) Matrix metalloproteinase 2 is asso- ciated with changes in steroid hormones in the sera and peritoneal fluid of patients with endometriosis. Fertility Sterility 81: 1235-1239. Itoh F, Komohara Y , Takaishi K, Honda R, Tashiro H, Takeya M, Katabuchi H (2014) Interaction between peritoneal M2 macrophages and endometrial stromal cells in the development of endometriosis via Stat3 activation. Am J Reprod Immunol 71: 56-57. Iwabe T, Harada T (2014) Inflammation and Cytokines in Endometriosis. In: Endo- metriosis, pp. 87-106. Kim BG, Yoo JY , Kim TH, Shin JH, Langenheim JF, Ferguson SD, Fazleabas AT, Young SL, Lessey BA, Jeong JW (2015) Aberrant activation of signal transducer and activator of transcription-3 (STAT3) signaling in endometriosis. Human Reprod 30: 1069-1078. Kloosterman WP, Plasterk RH (2006) The diverse functions of microRNAs in animal development and disease. Devel Cell 11: 441-450. Laudanski P, Charkiewicz R, Kuzmicki M, Szamatowicz J, Charkiewicz A, Niklinski J (2013) MicroRNAs expression profiling of eutopic proliferative endometrium in women with ovarian endometriosis. Reprod Biol Endocrinol 11: 78. Malvezzi H, Aguiar VG, Paz CCPd, Tanus-Santos JE, Penna IAdA, Navarro PA (2013) Increased circulating MMP-2 levels in infertile patients with moderate and severe pelvic endometriosis. Reprod Sci 20: 557-562. Nothnick WB (2017) MicroRNAs and endometriosis: distinguishing drivers from passengers in disease pathogenesis. In: Seminars in Reproductive Medicine, Thieme Medical Publishers, pp 173-180. O’Connell RM, Rao DS, Chaudhuri AA, Baltimore D (2010) Physiological and pathological roles for microRNAs in the immune system. Nature Rev Immunol 10: 111-122. Shi X-Y , Gu L, Chen J, Guo X-R, Shi Y-L (2014) Downregulation of miR-183 inhibits apoptosis and enhances the invasive potential of endometrial stromal cells in endo- metriosis. Int J Mol Med 33: 59-67. Tokushige N (2014) Role of nerve fibres in endometriosis. In: Endometriosis, pp. 191-211. Tsai E-M, Wang Y-S, Lin C-S, Lin W-Y , Hsi E, Wu M-T, Juo S-HH (2013) A microRNA-520 mirSNP at the MMP2 gene influences susceptibility to endometri- osis in Chinese women. J Human Genet 58: 202-209. Vercellini P, Viganò P, Somigliana E, Fedele L (2014) Endometriosis: pathogenesis and treatment. Nature Rev Endocrinol 10: 261-275. Viganò P, Parazzini F, Somigliana E, Vercellini P (2004) Endometriosis: epidemi- ology and aetiological factors. Best Prac Res Clin Obstet Gynaecol 18: 177-200. Wang Z, Zhang H, Zhang P, Li J, Shan Z, Teng W (2013) Upregulation of miR-2861 and miR-451 expression in papillary thyroid carcinoma with lymph node metas- tasis. Med Oncol 30: 577. ORIGINAL ARTICLES Pharmazie 74 (2019) 249 Xu J, Wan X, Chen X, Fang Y , Cheng X, Xie X, Lu W (2016) miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6. Sci Rep 6: 28968. Yoo J-Y , Jeong J-W, Fazleabas AT, Tayade C, Young SL, Lessey BA (2016) Protein inhibitor of activated STAT3 (PIAS3) is downregulated in eutopic endometrium of women with endometriosis. Biol Reprod 95: 11, 11-17. Zhang Y , Zhang D, Wang F, Xu D, Guo Y , Cui W (2015) Serum miRNAs panel (miR- 16-2*, miR-195, miR-2861, miR-497) as novel non-invasive biomarkers for detec- tion of cervical cancer. Sci Rep 5: 17942. Zhu M, Che Q, Liao Y , Wang H, Wang J, Chen Z, Wang F, Dai C, Wan X (2015) Oncostatin M activates STAT3 to promote endometrial cancer invasion and angio- genesis. Oncol Rep 34: 129-138.