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

Abstract

Background: Homeobox (HOX) genes encode transcription factors, which regulate cell proliferation, differentiation, adhesion, and migration. The deregulation of HOX genes is frequently associated with human reproductive system disorders. However, knowledge regarding the role of HOX genes in human granulosa cells is limited.

Methods: To determine the role of HOXA7 in the regulation and associated mechanisms of cell proliferation in human granulosa cells, HOXA7 and epidermal growth factor receptor (EGFR) expressions were examined in primary granulosa cells (hGCs), an immortalized human granulosa cell line, SVOG, and a granulosa tumor cell line, KGN, by real-time PCR and Western blotting. To manipulate the expression of HOXA7, the HOXA7 specific siRNA was used to knockdown HOXA7 in KGN. Conversely, HOXA7 was overexpressed in SVOG by transfection with the pcDNA3.1-HOAX7 vector. Cell proliferation was measured by the MTT assay.

Results: Our results show that HOXA7 and EGFR were overexpressed in KGN cells compared to hGCs and SVOG cells. Knockdown of HOXA7 in KGN cells significantly decreased cell proliferation and EGFR expression. Overexpression of HOXA7 in SVOG cells significantly promoted cell growth and EGFR expression. Moreover, the EGF-induced KGN proliferation was abrogated, and the activation of downstream signaling was diminished when HOXA7 was knocked down. Overexpression of HOXA7 in SVOG cells had an opposite effect.

Conclusions: Our present study reveals a novel mechanistic role for HOXA7 in modulating granulosa cell proliferation via the regulation of EGFR. This finding contributes to the knowledge of the pro-proliferation effect of HOXA7 in granulosa cell growth and differentiation.

Figure 1

HOXA7 and EGFR expression in human granulosa cells. Expression of HOXA7 and EGFR was detected in primary hGCs, the immortalized human granulosa cell line, SVOG, and the granulosa tumor cell line, KGN, cells by both real-time PCR and Western blotting. (A) Real-time PCR results of relative HOXA7 mRNA expression. (B) The representative autoradiographs of HOXA7 detected by Western blotting (indicated by arrow). (C) Real-time PCR results of relative EGFR mRNA expression. (D) The representative autoradiographs of EGFR detected by Western blotting. The data derived from at least three separate sets of experiments were standardized to the corresponding control, and the statistical results are presented in the column graphs. a, P < 0.05 compared with hGC; b, P < 0.05 compared with SVOG.

Figure 2

HOXA7 regulates granulosa cell proliferation. KGN cells were transiently transfected with scrambled siRNA or HOXA7 siRNA and the effect of HOXA7 knockdown was verified at the mRNA level by real-time PCR (A) and protein level by Western blotting (B). (C) After 36 h of transfection, KGN cells were reseeded into a 96-well plate and the cell viability was detected by MTT assay. SVOG cells were transiently transfected with control vector or HOXA7 plasmid and the effect of HOXA7 overexpression was verified at the mRNA level by real-time PCR (D) and protein level by Western blotting (E). (F) After 36 h of transfection, SVOG cells were reseeded into a 96-well plate and the cell viability was detected by MTT assay. The data derived from at least three separate sets of experiments were standardized to the corresponding control. a, P < 0.05 compared with control siRNA or control vector.

Figure 3

HOXA7 regulates EGFR expression in granulosa cells. KGN cells were transiently transfected with scrambled siRNA or HOXA7 siRNA. Expression of EGFR was detected at the mRNA level by real-time PCR (A) and protein level by Western blotting (B). SVOG cells were transiently transfected with control vector or HOXA7 plasmid and expression of EGFR at the mRNA level was detected by real-time PCR (C) and at the protein level by Western blotting (D). The data derived from at least three separate sets of experiments were standardized to the corresponding control, and the statistical results are presented in the column graphs. a, P < 0.05 compared with control siRNA or control vector.

Figure 4

EGF induces granulosa cell proliferation via the activation of the MAPK and PI3K/Akt pathways. KGN and SVOG cells were serum starved for 12 h and incubated in serum-free medium containing various concentrations of EGF and/or AG1478 for various periods of time. KGN cells (A) and SVOG cells (C) were left untreated (Control) or treated with increasing concentrations of EGF and/or AG1478 for 48 h. The viability was measured by the MTT assay. The data derived from at least three separate sets of experiments were standardized to the corresponding control, and the statistical results are presented in the column graphs. a, P < 0.05 compared with control; b, P < 0.05 compared with treatment with EGF 100 ng/mL alone. KGN cells (B) and SVOG cells (D) were treated with 100 ng/mL EGF for 5 min, 10 min, 30 min, 1 h, 3 h or were untreated (Control). The phosphorylation of ERK1/2 or Akt was determined by Western blotting with specific antibodies.

Figure 5

EGF-induced granulosa cell proliferation and activation of downstream signaling are modulated by the expression of HOXA7. KGN cells were transiently transfected with scrambled siRNA or HOXA7 siRNA. SVOG cells were transiently transfected with control vector or HOXA7 plasmid. KGN cells (A) and SVOG cells (C) were reseeded into a 96-well plate after 36 h of transfection. Cells were serum starved for 12 h and incubated in serum-free medium containing 100 ng/mL EGF and/or AG1478, or were left untreated (Control) for 48 h. Cell viability was measured by the MTT assay. KGN cells (B) and SVOG cells (D) were serum starved for 12 h after 36 h transfection and incubated in serum free medium containing 100 ng/mL EGF and/or AG1478, or left untreated (Control) for 10 min. The phosphorylation of ERK1/2 or Akt was determined by Western blotting with specific antibodies. The data derived from at least three separate sets of experiments were standardized to the corresponding control, and the statistical results are presented in the column graphs. a, P < 0.05 compared with control; b, P < 0.05 compared with control siRNA or control vector transfection and EGF treatment. c, P < 0.05 compared with EGF treatment alone.