Targeting of syndecan-1 by micro-ribonucleic acid miR-10b modulates invasiveness of endometriotic cells via dysregulation of the proteolytic milieu and interleukin-6 secretion  Cornelia Schneider, M.Sc., Nadja Kässens, M.D., Burkhard Greve, Ph.D., Hebatallah Hassan, M.Sc., Andreas N. Schüring, M.D., Anna Starzinski-Powitz, Ph.D., Ludwig Kiesel, M.D., Ph.D., Daniela G. Seidler, Ph.D., Martin Götte, Ph.D.  Fertility and Sterility  Volume 99, Issue 3, Pages 871-881.e1 (March 2013) DOI: 10.1016/j.fertnstert.2012.10.051 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 The siRNA knockdown of siSDC1 results in decreased invasiveness of 12Z cells. Control = control siRNA-treated cells; siSDC1 = Syndecan-1 siRNA–treated cells. (A) Quantitative PCR confirmation of successful siRNA-mediated knockdown of SDC1 mRNA expression 48 hours after siRNA transfection. Syndecan-1 expression was normalized to 18S rRNA expression (n ≥ 3, *P<.05). (B) Confirmation of siRNA-mediated SDC1 protein down-regulation by immunofluorescence microscopy. Only residual SDC1 staining in the Golgi-apparatus is observed 72 hours after siRNA-mediated depletion. (C) Confirmation of SDC1 siRNA knockdown by Western blotting (72 hours after transfection). Tubulin was used as a loading control. (D) Cell viability (MTT) assay. The siRNA depletion of SDC1 did not significantly affect cell viability (n = 3). (E) The siRNA depletion of SDC1 leads to a substantial inhibition of Matrigel invasiveness. See Materials and Methods for assay details. Left: Quantitative analysis of cell numbers invading Matrigel-coated filter membranes (*P<.05, n ≥ 3). Right: Representative micrographs of stained filter membranes. Small circles represent membrane pores, whereas transmigrated invasive cells show irregularly shaped dark grey staining. Fertility and Sterility 2013 99, 871-881.e1DOI: (10.1016/j.fertnstert.2012.10.051) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The siRNA knockdown of SDC1 in the 12Z endometriotic cell line results in reduced matrix metalloproteinase expression and activity, and reduced IL-6 secretion. 12Z cells were transfected with SDC1 siRNA or a negative control siRNA and cultured for 48 hours (qPCR analysis) or 72 hours (protein analyses), respectively. (A) Quantitative PCR analysis reveals significant down-regulation of MMP-9 in SDC1-depleted cells. PLAU = urokinase-type plasminogen activator (*P<.05, n = 3). (B) Gelatin zymography demonstrates reduced MMP-2 activity in the conditioned media of SDC1-depleted 12Z cells relative to controls. Recombinant MMP-2 was used as a positive control. Matrix metalloproteinase-2 activity staining is visible as a clear band against a background of Coomassie Blue–stained gelatin substrate after sodium dodecyl sulfate–polyacrylamide gel electrophoresis. (C) Western blot analysis reveals up-regulation of the protease inhibitor PAI-1 in SDC1-depleted cells. (D) Syndecan-1 siRNA-treated cells show decreased secretion of IL-6 into conditioned media relative to control cells, as demonstrated by ELISA (*P<.05, n ≥ 3). Fertility and Sterility 2013 99, 871-881.e1DOI: (10.1016/j.fertnstert.2012.10.051) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Syndecan-1 is a regulatory target of microRNA miR-10b in 12Z endometriotic cells and primary eutopic endometrial stroma cells. Cells were transfected with pre-miR-10b or a negative control miRNA and cultured for 48 hours before further analysis. (A) Quantitative PCR confirms successful up-regulation of miR-10b expression upon transient transfection of 12Z cells with miR-10b precursors, showing a dose response (*P<.05, **P<.01, n = 3). (B) Quantitative PCR analysis reveals a significant inhibitory effect of miR-10b on SDC1 expression in 12Z cells. Expression of the predicted targets F11R (JAM-A), KLF4, LEFTY2, and NOTCH4 was not significantly altered (*P<.05, n ≥ 9). (C) Quantitative PCR analysis reveals a significant inhibitory effect of miR-10b on SDC1 expression in primary eutopic endometrial stroma cell cultures from two endometriosis patients. Each patient was normalized to self as control. Dark grey bars = patient LW, light grey bars = patient PK (*P<.05, n = 4, paired t test). (D) The 3′ UTR of SDC1 is a target for transcriptional regulation by miR-10b, as determined by luciferase assay. 12Z cells were transfected with a plasmid expressing firefly luciferase (hLuc) under the control of an SV40 enhancer and the 3′ UTR of human SDC1, and renilla luciferase under constitutive control of the cytomegalovirus promoter (17). Cells were cotransfected with a control pre-miR or pre-miR-10b and simultaneously assayed for activity of both luciferases 72 hours after transfection. miR-10b transfection induced a significant decrease in SDC1-specific normalized firefly luciferase activity (n = 3, ***P<.001). (E) Flow cytometric analysis reveals down-regulation of SDC1 protein expression in 12Z cells upon transfection with miR-10b precursor molecules. Each plot shows MS-IgG-PC5 isotype-control antibody (dotted line) and MAH-CD138-PC5- (SDC1)-stained cells (solid line). The x axis represents the fluorescence intensity and the y axis the number of cells with a given fluorescence. Thus, the higher the peak, the more cells show a specific fluorescence, and the more the peak moves to the right part of the x axis, the higher is the fluorescence intensity. To quantify SDC1 (CD138)-specific fluorescence, a gate was set coming from the end of the isotype control peak (unspecific fluorescence) to the end of the x axis (specific fluorescence area), and the mean fluorescence was determined over the whole range. The mean SDC1 (CD138)-specific fluorescence was lower in the miR-10b precursor molecule transfected 12Z cells (2.3 SD 0.0) than in the control treated cells (4.3 SD 0.7). The median fluorescence intensity is given for each peak. (F) Immunofluorescence microscopy reveals down-regulation of SDC1 protein expression upon miR-10b precursor transfection of 12Z cells. Fertility and Sterility 2013 99, 871-881.e1DOI: (10.1016/j.fertnstert.2012.10.051) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 miR-10b up-regulation inhibits invasiveness of 12Z endometriotic cells in a SDC1-dependent manner. 12Z cells were transfected with pre-miR-10b or a negative control miRNA and cultured for 48 hours before further analysis. (A) Ectopic miR-10b expression moderately affects 12Z cell viability as determined by MTT assay (*P<.05, n = 3). (B) Up-regulation of miR-10b inhibits invasiveness of 12Z cells. Matrigel invasion assays were performed 72 hours after transfection with a control miR or a miR-10b precursor (n ≥ 3,∗*P<.01). Cotransfection of miR-10b and an Sdc1 construct lacking the endogenous 3′ UTR (18) abolishes the invasion-inhibiting effect of miR-10b, demonstrating involvement of miR-dependent SDC1 targeting in the invasion phenotype. (C) Western blot analysis demonstrates that miR-10b up-regulation reduces responsiveness of 12Z cells to HGF-induced MAPK activation. miR-10b- or control miRNA–transfected cells were serum-starved for 24 hours and stimulated with or without 40 ng/mL HGF for 30 minutes, 72 hours after transfection. Cell extracts were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blotting using antibodies specific for phosphorylated and total forms of p44/42 MAPK. Tubulin served as a loading control. (D) Hypothetical model of miR-10b–dependent regulation of SDC1 expression as a molecular mechanism that may promote the pathogenesis of endometriosis at multiple levels. The model is based on the results of this study and literature data. miR-10b down-regulation in endometriosis (9) promotes endometriotic cell proliferation via an unknown SDC1-independent mechanism. Down-regulation of miR-10b results in increased SDC1 expression, which is associated with increased IL-6 secretion and possibly enhanced signaling (19), thus promoting inflammatory processes in endometriosis (38–41). Syndecan-1 enhances growth factor activation of the MAPK pathway (17, 18, 21). Consequently, transcription factors (TF) are activated that up-regulate expression of MMPs and down-regulate the proteinase inhibitor PAI-1, thus driving an invasive phenotype of endometriotic cells. miR-10b–dependent regulation of HOXD10, and associated transcriptional changes in RHO C expression (47), may partially counteract this process. Fertility and Sterility 2013 99, 871-881.e1DOI: (10.1016/j.fertnstert.2012.10.051) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 (A) Dose-dependent silencing effect of SDC1 siRNA in 12Z cells. 12Z cells were transfected with 0 nM, 0.5 nM, 5 nM, or 20 nM SDC1 siRNA, and SDC1 expression was subsequently assayed by qPCR as described in Materials and Methods and Figure 1A of the main manuscript (n ≥ 4, *P=.06, *P<.05). (B) Nonsignificant changes in gene expression after SDC1 siRNA knockdown in 12Z cells. 12Z cells were transfected with SDC1 siRNA or a negative control siRNA and cultured for 48 hours. Quantitative PCR analysis of several genes potentially related to SDC1 function does not reveal significant changes in gene expression (n = 3). (C) Basal expression levels of SDC1 mRNA and miR-10b as determined by qPCR reveal a reduced expression of SDC1 (*P=.06), but no differences in miR-10b expression between 12Z endometriotic epithelial cells and primary eutopic stromal cells from endometriosis patients (pooled data from two patients) (n ≥ 3). (D) Quantitative PCR analysis of the influence of HGF on SDC1 and miR-10b expression in 12Z cells. 12Z cells were serum-starved for 24 hours and stimulated with or without 40 ng/mL HGF for 30 minutes or 5 hours, respectively, 72 hours after transfection, followed by qPCR analysis. Hepatocyte growth factor has no influence on SDC1 or miR-10b expression after 30 minutes' stimulation (compared with signaling data in Fig. 4C of the main text); however, miR-10b expression was significantly reduced, by >50%, after 5 hours' HGF stimulation (n ≥ 4, *P<.05). Fertility and Sterility 2013 99, 871-881.e1DOI: (10.1016/j.fertnstert.2012.10.051) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions