MicroRNA miR-145 inhibits proliferation, invasiveness, and stem cell phenotype of an in vitro endometriosis model by targeting multiple cytoskeletal elements and pluripotency factors  Marlene Adammek, B.Sc., Burkhard Greve, Ph.D., Nadja Kässens, M.D., Cornelia Schneider, M.Sc., Kathrin Brüggemann, B.Sc., Andreas N. Schüring, M.D., Anna Starzinski-Powitz, Ph.D., Ludwig Kiesel, M.D., Ph.D., Martin Götte, Ph.D.  Fertility and Sterility  Volume 99, Issue 5, Pages 1346-1355.e5 (April 2013) DOI: 10.1016/j.fertnstert.2012.11.055 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 miR-145 overexpression results in an inhibition of proliferation and Matrigel invasiveness of 12Z endometriotic cells (12Z) and in reduced proliferation of primary eutopic stromal cells. (A) Quantitative RT-PCR confirmation of miR-145 overexpression 48 hours after miR-145 precursor transfection of 12Z cells. miR-145 expression is normalized to RNU6B expression. Treatment with an miR-145–specific anti-miR inhibitor did not result in altered miR-145 expression, because the inhibitory mechanism is based on competitive binding. ***P<.001, error bars = SEM, n = 3. (B) Overexpression of miR-145 leads to a substantial inhibition of Matrigel invasiveness of 12Z cells. ***P<.001, error bars = SEM, n ≥ 8. (C–E) MTT cell proliferation assay. (C) 12Z cells transfected with miR-145 precursors, a control miR precursor, or an anti–miR-145 inhbitor. Forty-eight hours after transfection, 10,000 cells were allowed to proliferate for 24 hours, and an MTT assay was then carried out for 24 hours as described in Materials and Methods. miR-145 overexpression induced a significant inhibition of cell proliferation. *P<.05, error bars = SEM, n = 10. (D) Primary eutopic endometrial stroma cells derived from endometriosis patients were transfected and subjected to an MTT assay as described in (C). miR-145 overexpression induced a significant inhibition of cell proliferation. *P<.05, error bars = SEM, n = 3. (E) Primary ectopic endometrial stroma cells derived from deep infiltrating endometriotic lesions were transfected and subjected to an MTT assay as described in (C). miR-145 overexpression induced a significant inhibition of cell proliferation. *P<.05, error bars = SEM, n = 3. See Supplementary Table 1 for patient characteristics. (F) Cell cycle analysis of 12Z cells subjected to control or miR-145 precursor transfection. miR-145 increases the proportion of cells in the G2/M phase. Fertility and Sterility 2013 99, 1346-1355.e5DOI: (10.1016/j.fertnstert.2012.11.055) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Up-regulation of miR-145 reduces putative stem cell pools in the 12Z endometriotic cell line. 12Z cells were transfected with pre–miR-145, or a negative control miRNA, cultured for 72 hours, and subjected to analysis of surrogate parameters of stem cell activity. (A) Side population analysis. Transfected 12Z cells were incubated with the Hoechst 33342 dye in the presence or absence of 50 μM verapamil, followed by flow cytometry. Gate R2 shows SP cells. (B) Identification of putative endometriotic stem cells displaying ALDH activity. Transfected 12Z cells incubated with fluorescent ALDH substrate in the presence or absence of the inhibitor diethylaminobenzaldehyde, followed by flow cytometric analysis. Data are representative of three independent experiments, which yielded comparable results. Fertility and Sterility 2013 99, 1346-1355.e5DOI: (10.1016/j.fertnstert.2012.11.055) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Ectopic expression of miR-145 leads to a targeting of cytoskeletal elements and stemness-associated mRNAs. 12Z cells (A–F), primary eutopic endometrial stroma cells (G), and primary ectopic endometrial stroma cells (H) were transfected with pre–miR-145, a negative control miRNA, or an miR-145–specific anti-miR inhibitor (12Z only) and cultured for 48 hours before further analysis. Please note different scale of the y axis in individual panels. In 12Z cells, qRT-PCR analysis reveals a significant inhibitory effect of miR-145 on FASCIN1 (A), SOX2 (B), MSI2 (C), SERPINE1 (D), and JAM-A (E) expression. (F) Western blot analysis of differentially regulated JAM-A, fascin, SERPINE1/PAI-1, and c-Myc protein expression in miR-145 vs. control transfected 12Z cells. Extracts of cells transfected as described in Figure 1 were analyzed by Western blotting. Stripped blot membranes were reprobed with tubulin antibodies as a loading control. miR-145 up-regulation strongly inhibits JAM-A and fascin expression and leads to a moderate, yet reproducible inhibition of SERPINE1/PAI-1 expression. Expression of c-Myc is not altered in 12Z cells. Anti–miR-145 treatment had no clear effects. Representative blots of three individual experiments. (G, H) Quantitative PCR analysis of miR-145 target gene expression in primary eutopic endometrial stroma cells of three endometriosis patients (G) and/or primary ectopic stroma cells derived from endometriotic lesions of four patients (H) indicates concordant and significant down-regulation of FASCIN-1, SOX2, and MSI2 expression by miR-145. Data are expressed as fold change of miR-145 vs. control precursor transfected cells. All panels: error bars = SEM, ***P<.001, *P<.05, n ≥ 8 (D, E), n ≥ 4 (A–C, H), n = 3 (G). Fertility and Sterility 2013 99, 1346-1355.e5DOI: (10.1016/j.fertnstert.2012.11.055) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Dysregulation of miR-145 expression promotes the pathogenesis of endometriosis by modulating targets regulating proliferation, invasiveness, and stem cell properties. Model based on the current in vitro study and literature data (see Discussion for details). miR-145 is dysregulated in endometriotic lesions and eutopic endometrium compared with healthy endometrium (15–18). Changes in miR-145 expression affect posttranscriptional regulation of target gene expression in the 12Z cell line derived from an ectopic endometriotic lesion and in primary eutopic and ectopic endometrial stroma cells: Fascin-1, additional cytoskeletal elements, JAM-A, and PAI-1 modulate invasiveness (11, 12, 20), thus facilitating establishment of the ectopic lesion and cell migration to the ectopic site. Dysregulated expression of SOX2, MSI2, and additional pluripotency factors promotes stem cell function (6, 21, 30, 31), which may enhance unlimited proliferative potential and high differentiation potential at the ectopic site. In addition, Fascin-1, SOX2, MSI2, and OCT4 modulate cell proliferation (30, 38, 39, 43), thus promoting growth of the endometriotic lesion. Fertility and Sterility 2013 99, 1346-1355.e5DOI: (10.1016/j.fertnstert.2012.11.055) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplementary Figure 1 Alignment of the miR-145 seed sequence and the corresponding 3' UTR sequences in target mRNAs. Putative target sites were calculated using the miRanda algorithm according to the microRNA.org database (1): F11R (JAM-A), FSCN1 (Fascin-1), PODXL (Podocalyxin), SERPINE1/PAI-1, SOX2, and KLF4 are among the predicted targets of miR-145. 1. Betel D, Wilson M, Gabow A, Marks DS, Sander C. The microRNA.org resource: targets and expression. Nucleic Acids Res 2008;36(Database Issue):D149–53. Fertility and Sterility 2013 99, 1346-1355.e5DOI: (10.1016/j.fertnstert.2012.11.055) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Supplementary Figure 2 miR-145–dependent changes in gene expression in the endometriotic cell line 12Z and in primary eutopic and ectopic endometrial stroma cells. 12Z cells (A–F), primary eutopic endometrial stroma cells of three endometriosis patients (G), and primary ectopic endometrial stroma cells derived from endometriotic lesions of four patients (H) were transfected with pre-miR-145, a negative control miRNA, or an miR-145–specific anti-miR inhibitor (12Z cells only) and cultured for 48 hours before further analysis. In 12Z cells (A–G), qRT-PCR analysis reveals a significant inhibitory effect of miR-145 on PODXL (A), OCT-4 (B), and KLF4 (C) expression. (D) NODAL expression in 12Z cells is significantly inhibited upon miR-145 inhibition, whereas pre–miR-145 transfection leads to a significant up-regulation of ACTG2 (E) and TGLN (F). (G) The 3' UTR of JAM-A is a target for transcriptional regulation by miR-145 in 12Z cells, 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 JAM-A, and renilla luciferase under constitutive control of the cytomegalovirus promoter (1). Cells were cotransfected with a control pre-miR or pre–miR-145 and simultaneously assayed for activity of both luciferases 72 hours after transfection. miR-145 transfection induced a significant decrease in JAM-A–specific normalized firefly luciferase activity (n = 3, **P<.01). (H, I) Quantitative PCR analysis of miR-145 target gene expression in primary eutopic endometrial stroma cells from three ASRM III endometriosis patients (H) and in primary ectopic endometrial stroma cells derived from endometriotic lesions of four patients with deep infiltrating endometriosis. Data are expressed as fold change of miR-145 vs. control precursor transfected cells. All panels: error bars = SEM, ***P<.001, *P<.05; n ≥ 8 (A, C, E), n ≥ 4 (B, D, F, G, I), n = 3 (H). 1. Ramón LA, Gilabert-Estellés J, Cosín R, Gilabert J, España F, Castelló R, et al. Plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphism and endometriosis. Influence of PAI-1 polymorphism on PAI-1 antigen and mRNA expression. Thromb Res 2008;122:854–60. Fertility and Sterility 2013 99, 1346-1355.e5DOI: (10.1016/j.fertnstert.2012.11.055) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions