Isolation and culture of human endometrial stromal cells
HESCs were isolated from endometrial tissue obtained via endometrial biopsy from normal fertile women with regular menstrual cycles (n = 25) and eutopic endometrial stromal cells were isolated from eutopic endometrial of patients with pelvic endometriosis (n = 7). All the endometriosis patients were diagnosed by the laparoscopy and the age of the patients were 25 to 35 years old. The Drum Tower Hospital Research and Ethics Committee approved this study, and all of the patients gave their informed consent. The tissues were immediately placed into culture medium and processed according to Sun et al. , with minor modifications. First, the endometrial tissues were minced and enzymatically digested with 0.1% (w/v) collagenase I (Worthington, Freehold, NJ, USA) for 30 min at 37°C. Next, the digested tissues were filtered through 30 μm-sieve gauze to separate the stromal cells from the glands. The endometrial stromal cells were maintained in DMEM/F12 supplemented with 10% (v/v) FBS, 50 IU/mL of penicillin and 50 μg/mL of streptomycin (Gibco BRL/Invitrogen, Carlsbad, CA, USA), seeded into culture dishes and incubated at 37°C in 5% CO2. The cultured stromal cells were 95% pure, as determined by vimentin staining.
An adenovirus construct bearing the human full-length CAPN 7 gene (Ad-Flag-CAPN 7) was produced using AdMax (Microbix, Mississauga, Ontario, Canada). An adenovirus bearing LacZ (Ad-LacZ) was obtained from BD Biosciences Clontech (Palo Alto, CA, USA). The viruses were packaged and amplified in HEK293A cells and purified using CsCl banding, followed by dialysis against 10 mmol/L Tris-buffered saline with 10% (w/v) glycerol. Titration was performed in HEK293A cells with the Adeno-X Rapid Titer kit (BD Biosciences Clontech) according to the manufacturer’s instructions. HESCs were infected with Ad-LacZ or Ad-Flag-CAPN 7 at an MOI of 50.
siRNA knockdown assays
A pair of small interfering (si)RNA oligonucleotides specific for human CAPN 7 (sense strand: 5′-CAUUAGUGGUUUCUCAAUAdTdT-3′ and 3′-dTdT GUAAUCACCAAAGAGUUAU-5′) and a pair of control siRNA oligonucleotides were synthesized by RIBOBIO (Guangzhou, China). HESCs were grown to 70-80% confluence and transfected with siRNAs with the SuperFectTMII (Pufei Biotech, Shanghai, China) transfection reagent at a final concentration of 50 nM according to the manufacturer’s recommendations.
Cell migration and invasion assays
The wound and invasion assays were performed according to Rai et al. , with minor modifications. For the scratch wound assay, cells were starved in DMEM/F12 plus 2.5% (v/v) FBS for 24 hours before the assay and the cells were maintained in DMEM/F12 plus 2.5% (v/v) FBS for the entire experiment. When the cells reached 80-90% confluence, the hESCs were infected with adenovirus at 50 MOI or transfected with 50 nM siCTL or 50 nM siCAPN 7. The confluent cell monolayer was wounded with a 200 μL plastic cell scraper at the 0 h time point (immediately after siRNA transfection or 6 h after adenovirus infection). Cell migration was evaluated at the wound front at 0, 24, 48 and 72 h after wounding. For the matrigel-coated invasion chamber assay, cells were also starved in DMEM/F12 plus 2.5% (v/v) FBS for 24 hours and then infected with Ad-LacZ or Ad-Flag-CAPN 7 at an MOI of 50 or transfected with siCTL or siCAPN 7 at a final concentration of 50 nM. Polycarbonate membrane filters (8 μm pore size) (Millipore Corporation, Billerica, MA, USA) were pre-coated with matrigel. Next, the cells (5 × 104 cells per chamber) in 100 μL DMEM/F12 plus 0.1% (w/v) BSA were added to the top chambers. The bottom chambers were filled with 700 μL DMEM/F12 supplemented with 10% (v/v) FBS and then incubated at 37°C for 72 h. Then, the invasion cells were fixed, dyed and counted. For inhibitor experiments, OA-Hy (Millipore Corporation, Billerica, MA, USA) was added at a concentration of 20 μM 1 h before adenovirus infection; the OA-Hy concentration was maintained throughout the experiment. All the experiments were performed at three times and each experiment was performed with cells isolated from three patients (hESC were isolated from normal fertile women and the cultured stromal cells were 95% pure, n = 9).
Total proteins were extracted and analyzed via western blotting as described previously . HESCs and endometrium were lysed in lysis buffer (50.0 mmol/L Tris, pH 7.6, 150.0 mmol/L NaCl, 0.1% (w/v) SDS, 1.0% (w/v) NP-40, protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA)). The protein concentrations in the total lysates were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (30 μg) were separated on 10% (v/v) sodium dodecyl sulfate (SDS)–polyacrylamide gel by electrophoresis for 1.5 h, then transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA) and probed with the following antibodies as appropriate: anti-CAPN 7 (1:1000 dilution; Santa Cruz Biotechnology, CA, USA, SC-50501), anti-MMP-2 (1:1000 dilution; Bioworld Technology, MN, USA, BS1236), anti-Flag-HRP (1:2000 dilution; Sigma, St. Louis, MO, USA, A8592) and anti-β-actin (1:5000 dilution; Abcam, Cambridge, MA, USA, AP0060). An enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ, USA) was used to visualize the blots.
Precipitations were performed as previously described . Briefly, 600 μg of proteins was immunoprecipitated with anti-rabbit IgG or anti-AP2α (Bioworld Technology, MN, USA, BS1015) at 4°C for 12 h. The washed precipitates were detected by western blotting as described above.
Quantitative real-time PCR
Total RNAs were isolated using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Two micrograms of total RNA were subsequently reverse transcribed in a total volume of 25 μL at 37°C for 1 hour to produce cDNA, and SYBR Green fluorescence was measured as described previously . The reactions were performed using a MyiQ Single Color Real-time PCR detection system (Bio-Rad) for 40 cycles (95°C for 15 s, 55°C for 30 s, and 72°C for 30 s) after an initial 3 min incubation at 95°C. The specific primers used for 18S rRNA detection were 5′-CGGCTACCACATCCAAGGAA-3′ and 5′-CTGGAATTACCGCGGCT-3′, the CAPN 7 primers were 5′-ATGGTGTCCCAAGAAAGGTG-3′ and 5′-TGGTATCCAGCCAGTCAGTG-3′, the MMP-2 primers were 5′-ACATCAAGGGCATTCAGGAG-3′ and 5′-ATCTCACCACGGATCTGAAC-3′, and the TIMP-2 primers were 5′-CCAAGCAGGAGTTTCTCGAC-3′ and 5′-GACCCATGGGATGAGTGTTT-3′. The efficacy of CAPN 7, MMP-2 and TIMP-2 is 90.4%, 92.7% and 100.8% respectively and the expression level of each gene was normalized against the internal reference gene 18S to detect fold changes in expression. Next, melting curve and agarose gel electrophoresis analyses were used to confirm the specificity of the obtained PCR products and the real-time PCR results.
Gelatin zymography was performed as previously described with some modifications . Supernatants were collected from hESCs treated with adenovirus or siRNA for 48 h in DMEM/F12 plus 2.5% (v/v) c-FBS. Equal amounts of protein (10 μg) were separated on 10% (v/v) SDS-polyacrylamide gels that contained gelatin by electrophoresis for 4 h, then the gels were washed with 2.5% (v/v) Triton X-100 (2 × 20 min). Next, the gels were incubated in a post-electrophoretic buffer (50 mmol/L Tris, 5 mmol/L CaCl2, 200 mmol/L NaCl, and 3% (w/v) Brij-35) at 37°C for 36 h and stained with 0.125% (w/v) coomassie brilliant blue for 1 h. Finally, the gels were destained in 30% (v/v) methanol/10% (v/v) glacial acetic acid. Various MMPs were distinguished according to their molecular weights.
All experiments in this study were performed at least three times. Statistical analysis was performed with ANOVA, followed by the Student–Newman-Keulsmultiple comparisons test. P < 0.05 was considered statistically significant.