Homeobox A7 increases cell proliferation by up-regulation of epidermal growth factor receptor expression in human granulosa cells

Primary human granulosa cells (hGCs) were obtained with the approval of the Research Ethics Board of the University of British Columbia. hGC isolation from follicular aspirates collected during oocyte retrieval from women undergoing in vitro fertilization was performed as previously described [28]. In each well of six-well culture plates, 3 × 10⁵ viable cells were seeded and cultured in a humidified atmosphere of 5% CO₂-95% air at 37°C in 1:1 Dulbecco modified Eagle medium and Ham's F-12 medium (DMEM/F12; Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT, USA), 100 U/mL penicillin G and 0.1 mg/mL streptomycin sulfate (Invitrogen, Burlington, ON, CA). After 48 h, the hGCs were harvested from the plate for RNA or protein extraction.

KGN cells were maintained in 1:1 DMEM/F12, supplemented with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The SVOG cells were maintained in M199/MCDB105 (Invitrogen) supplemented with 10% FBS, 100 U/mL penicillin G, and 0.1 mg/mL streptomycin. In each well of six-well culture plates, 2 × 10⁵ viable KGN or SVOG cells were seeded, and cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂. The cells were sub-cultured when they reached about 90% confluence using a trypsin/EDTA solution (0.05% trypsin, 0.5 mM EDTA).

The monoclonal HOXA7 antibody, polyclonal EGFR antibody and polyclonal β-actin antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The monoclonal phospho-ERK1/2 (Thr202/Tyr204) antibody, polyclonal total ERK1/2 antibody, polyclonal phospho-Akt (Ser473) and polyclonal total-Akt antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Horseradish peroxidase-conjugated donkey anti-goat IgG was obtained from Santa Cruz. Human epidermal growth factor (EGF) and the EGFR inhibitor, AG1478, were obtained from Sigma.

KGN cells were cultured in growth medium as described above to achieve approximately 50-60% confluence at the time of transfection. Transfections were performed using a non-targeting negative control siRNA (Dharmacon, Chicago, IL, USA) or siRNAs against human HOXA7 (ON-TARGET plus SMART pool, Dharmacon) at a final concentration of 75 nM using Lipofectamine RNAiMAX (Invitrogen). Following transfection, the cells were directly processed for mRNA/protein extraction at different time points (24 h, 48 h and 72 h after transfection), or reseeded into new culture plates after 36 h of transfection for further cell viability analysis (24 h, 48 h, 72 h and 96 h after reseeding) or EGF treatment for various periods of time (48 h for cell viability analysis and 10 min for signaling pathway detection).

Full-length HOXA7 (917 bp) was amplified by RT-PCR using the Phusion RT-PCR Kit (New England BioLabs, Ipswich, MA, USA) and the following primers (5'-3'): TCA TTC CTC CTC GTC C and ATG AGT TCT TCG TAT TAT GAA CG. Gel-purified PCR product was cloned into the pcDNA3.1 (+) vector using the pCR- Blunt II-TOPO Expression Kit (Invitrogen) and confirmed by DNA sequencing (Child & Family Research Institute DNA Sequencing Core Facility, Vancouver, BC, CA). pcDNA3.1/CAT was used as a control vector. SVOG cells were cultured in growth medium as described above to achieve approximately 80-90% confluence at the time of transfection. Transient transfections were carried out using Lipofectamine 2000 Reagent (Invitrogen) following the manufacturer's protocol. After transfection, cells were directly processed for mRNA/protein extraction at different time points, or reseeded into new culture plates after 36 h of transfection for further cell viability analysis or EGF treatment for various periods of time, as described above.

Cell growth was evaluated via the 3-(4, 5-dimethyl thiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, 1 × 10⁴ cells were seeded in triplicate in 96-well plates. In some experiments, cells with or without HOXA7 transfection were treated with EGF, AG1478, or EGF and AG1478. At the indicated times, a final concentration of MTT at 500 μg/mL (Sigma) was added and the cells were incubated for an additional 4 h at 37°C. Following the incubation period, DMSO was added to dissolve the crystals. The absorbance was measured at 492 nm and was expressed as the relative proliferation rate.

At the end of the treatment period, the medium was removed from the culture plate and RNA was extracted using TRIzol (Invitrogen). RNA concentration was measured based on the absorbance at 260 nm. The isolated RNA was reverse transcribed into first-strand cDNA using M-MLV reverse transcriptase (Promega BioSciences, San Luis Obispo, CA, USA). The primers used for SYBR Green real-time RT-PCR were designed using Primer Express Software v2.0 (PerkinElmer Applied Biosystems, Foster City, CA, USA). The primers for HOXA7 are: sense, 5'-TAC CCC TGG ATG CGG TCT T-3'; and antisense, 5'-CAG GTA GCG GTT GAA GTG GAA-3'. The primers for EGFR are: sense, 5'-CAT GTC GAT GGA CTT CCA GA-3'; and antisense, 5'-GGG ACA GCT TGG ATC ACA CT-3'. The primers for GAPDH are: sense, 5'-ATG GAA ATC CCA TCA CCA TCT T-3'; and antisense, 5'-CGC CCC ACT TGA TTT TGG-3'. Real-time PCR was performed on the ABI PRISM^® 7300 Sequence Detection System according to the manufacturer's protocol (Applied Biosystems). Amplification specificity was determined using the melting curve. Analysis and quantification of the relative mRNA levels was detected using the comparative Ct method. All real-time experiments were run in triplicate, and a mean value was used for the determination of mRNA levels. Negative controls containing water were used in each experiment. Relative quantification of the mRNA levels for HOXA7 and EGFR in granulosa cells was performed using the comparative Ct method with GAPDH as an endogenous control. Changes following treatments were recorded as fold differences from the values in untreated controls at each time point, as appropriate, with the formula 2^-ΔΔCt.

After treatment, cells were washed twice with ice-cold PBS and harvested in ice-cold lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg/mL leupeptin) containing protease inhibitors (Sigma). The extract was centrifuged at 14,000 rpm for 15 min at 4°C to remove cellular debris. Protein concentrations were determined by the Bradford method (Bio-Rad Laboratories). SDS-PAGE was used to further separate 30 μg of protein sample, which was then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked for 1 h in Tris-buffered saline containing 0.01% Tween 20 with 5% non-fat dried milk and were incubated overnight at 4°C with the relevant primary antibodies. After washing, the membranes were incubated with the secondary antibody, a peroxidase-conjugated anti-IgG for 1 h. Immunoreactive proteins were detected using enhanced chemiluminescence reagents (Amersham Bioscience, Baied'Urfe, Quebec, CA). For the detection of antibody bound to HOXA7, we used the SuperSignal Femto West (Pierce Chemical. Rockford, IL, USA) reagent as described by the manufacturer. β-actin was used as the internal control.

Data are presented as the means ± SEM from at least three independent experiments. Data were analyzed by one-way ANOVA, followed by Tukey's multiple comparison tests if the overall P values were significant, using the computer software PRISM (GraphPad Software, San Diego, CA, USA). Data were considered significantly different from each other at P < 0.05.

Expression of HOXA7 was detected in hGCs, SVOG and KGN cells by both real-time PCR and Western blotting. The expression level of HOXA7 in the granulosa tumor cell line KGN was significantly higher than in primary granulosa cells and the immortalized human granulosa cell line, SVOG (Fig. 1, A and 1B). EGFR expression was also detected in all cell lines. Consistent with HOXA7, KGN cells expressed significantly higher levels of EGFR compared to hGCs and SVOG cells (Fig. 1, C and 1D).

To determine the functional role of endogenous HOXA7, we used siRNA to knockdown the expression of HOXA7 in KGN cells, which expressed high levels of HOXA7. HOXA7 knockdown was verified at the mRNA level by real-time PCR (Fig. 2, A) and at the protein level by Western blotting (Fig. 2, B). To examine the role of HOXA7 in granulosa cell proliferation, KGN cells transfected with HOXA7 siRNA or control siRNA were processed for the MTT assay. In response to HOXA7 knockdown, the proliferation rate of KGN cells was significantly decreased (Fig. 2, C).

To further determine the role of HOXA7 in human granulose cells, overexpression of HOXA7 was performed in the SVOG cell line, which expressed low levels of HOXA7. HOXA7 overexpression in SVOG cells was also verified at both the mRNA and protein levels by real-time PCR and Western blotting, respectively (Fig. 2, D and 2E). Overexpression of HOXA7 significantly increased cell proliferation in SVOG cells (Fig. 2, F).

The expression level of EGFR in granulosa cells after HOXA7 knockdown or overexpression was detected at both the mRNA level and protein level. Knockdown of endogenous HOXA7 in KGN cells significantly reduced EGFR expression, especially after 48 h of transfection (Fig. 3, A and 3B). Conversely, HOXA7 overexpression in SVOG cells significantly increased EGFR mRNA and protein levels, especially after 48 h of transfection (Fig. 3, C and 3D).

Cell viability assays showed that EGF promoted KGN and SVOG cell proliferation, especially at the concentration of 100 ng/mL (Fig. 4, A and 4C). Therefore, 100 ng/mL EGF was used in the subsequent experiments. The stimulatory effects of EGF could be abolished by the co-treatment with 1 μM of the specific EGFR inhibitor, AG1478 (Fig. 4, A and 4C). In addition, Western blotting results demonstrated that the treatment of both KGN and SVOG cells with EGF induced the phosphorylation of ERK1/2 and Akt, suggesting that the MAPK and PI3K/Akt signaling pathways play a role in mediating the induction of proliferation by EGF in the granulosa cell lines (Fig. 4, B and 4D).

Treatment with EGF significantly induced proliferation of KGN cells; however, this effect was abrogated when HOXA7 was knocked down by siRNA transfection (Fig. 5, A). In addition, the EGF-induced phosphorylation of Akt was diminished after HOXA7 knockdown. Quantitative analysis showed that, while the basal phosphorylation was not changed, the EGF-induced phosphorylation of Akt was significantly decreased (Fig. 5, B). In contrast, EGF-induced cell proliferation was enhanced in SVOG cells after HOXA7 overexpression, and this effect could be completely abolished by co-treatment with AG1478 (Fig. 5, C). Accordingly, the EGF-induced phosphorylation of Akt was significantly increased after HOXA7 overexpression, and this effect could also be abolished by treatment with AG1478. However, the knockdown or overexpression of HOXA7 did not significantly change the phosphorylation of ERK1/2 in either cell line (Fig. 5, B and 5D).