| Objective:To study the molecular mechanism of GPR30-EGFR signaling pathway in leiomyoma cells at bisphenol A exposure by detecting the GPR30 and EGFR changes. To provide the theory for prophylactic measures to reduce the incidence of uterine fibroids.Methods:1. Real-Time PCR method was applied for the detection of the GPR30 and EGFR mRNA expression in 15 leiomyomata and adjacent normal smooth muscles, whilst western blot assay was used for the detection of the GPR30 and EGFR protein expressions.2. The human uterine leiomyoma (hUL) cells were prepared by primary culture and subculture methods, which were identified to be smooth muscle cells by immunocytochemical staining with a monoclonal anti-a-smooth muscle actin antibody. Finally, 10μmol/L BPA was used to be the concentration for the next experiment. CCK-8 assay was used for the viability of humen leiomyoma cells in the 12 setting groups, including Group BPA(10μmol/L), Group Gl(GPR30 agonist) (1μmol/L),Group G15(GPR30 special inhibitor)(1μmol/L),Group ICI182780(ERantagonist)(1μmol/L),GroupAG1478(EGFRinhibitor)(10μmol/L),GroupPD980 59(MEKinhibitor)(10μmol/L),GroupG15+BPA(1μmol/L+10μmol/L),GroupICI182780+BPA( 1 μmol/L+10μmol/L),GroupAG 1478+BPA(10μmol/L+10μmol/L),GroupPD98059+BP(10μmo 1/L+10μmol/L) and control group (DMSO).3. Real-Time PCR method and western blot assay were used to test the expressions of GPR30 mRNA,EGFR mRNA,GPR30 protein,EGFR protein.4. Western bolt assay was used to test the expressions of c-fos, phospho-ERK1/2 proteins.Results1. The expressions of GPR30 and EGFR mRNA and protein in the tissue of leiomyomata are obviously higher than adjacent normal smooth muscle tissues.2. Primarily cultured hUL5-6 generation limited cells line was successfully established, which was identificated by the immunohistochemical staining.3. When compared with control group, there were significantly increasing proliferation rate of the hUL cells in BPA group (P＜0.05) and decreasing proliferation rate of hUL cells in G15、AG、PD groups in coexistence with BPA(P<0.05).4. Real-Time PCR method and Western bolt method study found that mRNA and protein expressions of GPR30 and EGFR were increased in the setting of BPA group、G1 group compared to control group (P<0.05) and decreased in the setting of G15 group^ AG group、PD group (P＜0.05). With the overlaid action of BPA, mRNA and protein expressions of GPR30 and EGFR were decreased in the setting of G15 group、AG group、PD group(P<0.05).5. When compared with control group, protein expressions of c-fos、P-ERK1/2 were significantly increased in Group BPA、Group G1 and decreased in Group G15、Group AG、 Group PD. With the overlaid action of BPA, mRNA and protein expressions of c-fos、 p-ERK1/2 were decreased in Group G15、Group AG、Group PD compared to isolated BPA group(P<0.05).Conclusions:1. There are growthing expressions of GPR30 and EGFR mRNA and protein in leiomyomata.2. BPA may be a promoter to the proliferation of uterine leiomyoma cells. The mechanism may result from GPR30-EGFR and MAPK/ERK/c-fos signaling pathway activation. |
