Decorin induced by progesterone plays a crucial role in suppressing endometriosis

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   Figure 1

   (A) Progesterone treatment promoted DCN mRNA expression in EMOsis cc/TERT cells and CRL-4003 cells. The mRNA expression of decorin was determined by real-time PCR from the total RNA obtained from CRL-4003 cells and EMOsis cc/TERT cells cultured with or without decorin or dienogest for 12 h. The mRNA levels of decorin were normalized to those of GAPDH mRNA. The data were processed by the comparative Ct method and expressed as a fold-increase relative to the basal transcription level in the control. Bars indicate the s.e.m. Significant differences are indicated by asterisks. *P<0.05, **P<0.01. (B) Progesterone treatment promoted the expression of decorin in EMOsis cc/TERT cells and CRL-4003 cells. CRL-4003 cells and EMOsis cc/TERT cells were harvested and used to prepare cell lysates after treatment with or without dienogest or progesterone for 24 h. The lysates were subjected to SDS–PAGE and blotted with anti-decorin (upper panel) or anti-β-actin (lower panel) antibodies. The lower panel shows the densitometric quantification of the western blot analysis normalized to β-actin expression and expressed as a fold-increase relative to the basal transcription level in the control. The mean ±s.d. of three determinations is shown. *P<0.05. (C) Progesterone treatment promoted the secretion of decorin in EMOsis cc/TERT cells and CRL-4003 cells. CRL-4003 cells and EMOsis cc/TERT cells were treated with dienogest at various concentrations for 48 h, and the concentrations of decorin produced by the EMOsis cc/TERT and CRL-4003 cells were measured using the Decorin (DCN) Human ELISA kit. The data are expressed as the mean±s.d. (N=5). * indicates a significant (P<0.05) difference compared with the untreated control group, while ⋇ indicates a significant (P<0.05) difference compared with untreated control EMOsis cc/TERT cells.
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   Figure 2

   (A) Progesterone treatment inhibits the proliferation of EMOsis cc/TERT cells and CRL-4003 cells in a decorin-dependent manner. CRL-4003 cells, EMOsis cc/TERT cells, and EMOsis cc/TERT cells transfected with the siRNA specific for decorin (si-DCN) were treated with dienogest at various concentrations with or without the decorin-neutralizing antibody (5 μg/ml) for 48 h, and the proliferation was measured by the MTS assay. (B) Decorin treatment inhibits the proliferation of EMOsis cc/TERT cells and CRL-4003 cells in a decorin-dependent manner. CRL-4003 cells and EMOsis cc/TERT cells were treated with decorin at various concentrations with or without a decorin-neutralizing antibody (5 μg/ml) for 48 h, and the proliferation was measured by the MTS assay. The data are expressed as the means±s.d. (n=5), and * indicates a significant (P<0.05) difference compared with the untreated control.
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   Figure 3

   Dienogest and progesterone play a crucial role in stimulating the DCN gene expression in EMOsis cc/TERT and CRL-4003 cells. (A) The half-PRE island is located in the Decorin promoter. The nucleotide sequence and putative regulatory elements of the 5′-flanking region of the human DCN gene are shown. The coding sequence of the human DCN gene is indicated by bold and capital letters. The blue underlined letters and red double underlined letters indicate the CAAT-box and TATA-box respectively. The large A residue at position +1 with an arrowhead above indicates the transcriptional initiation site. Boxed letters indicate the half-PRE sites. The arrows indicate the primer used for PCR amplification in the ChIP assay. (B) Progesterone and dienogest directly stimulated decorin gene expression. The ChIP assay, performed as described in the Materials and methods section, showed the expression of the decorin promoter in the control medium or progesterone or dienogest-stimulated EMOsis cc/TERT and CRL-4003 cells. An anti-PR antibody (Santa Cruz Biotechnology) was used for immunoprecipitation. The input DNA represents PCR products from chromatin pellets before immunoprecipitation. IgG was used as the negative control, and normal rabbit anti-IgG antibodies were used. The PCR primer was designed as described in the Materials and methods section.
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   Figure 4

   Dienogest induces cell cycle arrest in EMOsis cc/TERT cells and CRL-4003 cells by promoting p21 production and affects MET expression. (A) Inhibition of cell cycle progression by decorin and dienogest. EMOsis cc/TERT cells and CRL-4003 cells were exposed to 100 or 500 nmol/l dienogest or 3 or 6 μg/ml decorin overnight, or were left untreated, and then processed for PI flow cytometry. The cell cycle characteristics (G0–G1: S: G2–M%) of EMOsis cc/TERT cells were as follows: control (52.64±0.67: 29.45±0.92: 17.91±1.08), 100 nM dienogest (66.39±0.18*: 19.33±0.36*: 14.28±1.81), 500 nM dienogest (80.38±0.57*: 12.14±0.32*: 7.48±0.37*), 3 mg/ml decorin (67.59±0.95*: 19.8±0.04*: 12.61±0.83), and 6 mg/ml decorin (77.21±0.26*: 14.51±0.64*: 8.28±0.56*) and CRL-4003 cells: control (57.23±0.10: 20.46±0.63: 22.31±0.71), 100 nM dienogest (66.57±0.38*: 15.23±0.09: 18.2±0.26), 500 nM dienogest (78.07±0.69*: 13.61±0.17*: 8.32±0.91*), 3 mg/ml decorin (65.05±0.50*: 15.22±0.75: 19.73±0.27), and 6 mg/ml decorin (75.31±0.47*: 15.28±1.08*: 9.41±0.29*). The data are expressed as the means±s.d. (N=5), and * indicates a significant (P<0.05) difference compared with the untreated control. The panel shows the proportion of cells in the G0/G1 phase (black), S phase (gray), and G2/M phase (white). (B) The expression of p21 was increased and the expression of MET was decreased by dienogest and progesterone in a decorin-dependent manner. Proteins were extracted from CRL-4003 cells and EMOsis cc/TERT cells after treatment with 100 nmol/l dienogest, 100 nmol/l progesterone, or 100 nmol/l dienogest with or without 5 μg/ml of a decorin-neutralizing antibody for 24 h. The western blot analysis was performed with anti-Met antibodies, anti-p21 antibodies, and anti-β-actin antibodies. The lower panel shows the densitometric quantification of the western blot analysis normalized to the β-actin expression and expressed as a fold-increase relative to the basal transcription level in the control. The mean±s.d. of three determinations is shown. *P<0.05 (C) Decorin and p21 expression is increased and MET expression is decreased by dienogest and progesterone in a decorin-dependent manner. Proteins were extracted from CRL-4003 cells, EMOsis cc/TERT cells, and EMOsis cc/TERT cells transfected with DCN siRNA cultured with or without progesterone or dienogest for 24 h. The western blot analysis was performed with anti-decorin antibodies, anti-p21 antibodies, anti-Met antibodies, and anti-β-actin antibodies. The lower panel shows the densitometric quantification of the western blot analysis normalized to the β-actin expression and expressed as a fold-increase relative to the basal transcription level in the control. The mean±s.d. of three determinations is shown. *P<0.05
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   Figure 5

   Progesterone treatment promoted the expression of decorin in ovarian endometrioma. (A) DCN mRNA expression in patients with ovarian endometrioma. A total of 50 cases were selected for the study, and RNA was extracted from surgically obtained endometrioma tissue samples from the control group (n=25) and dienogest group (n=25). The mRNA levels of decorin were measured by real-time PCR and normalized to those of GAPDH mRNA. The data were processed by the comparative Ct method and expressed as a fold-increase relative to the basal transcription level in the control. Bars indicate the s.e.m. Significant differences are indicated by an asterisk. *P<0.05 (B) Decorin and MET expression in ovarian endometrioma. Representative examples of immunohistochemical sequential sections stained with decorin and MET among the ovarian endometrioma samples obtained from endometriosis patients who underwent oophorectomy are shown. A representative section from the no-preoperative-treatment control group is on the left, and a representative section from the dienogest preoperative treatment group is on the right. The black filled arrows indicate the epithelial cells and the white filled arrows indicate the stromal cells.

• © 2014 The authors