Expression And The Signaling Transduction Pathway Regulation Of PTEN Gene In Endometriosis

Nowadays, the pathogenesis of endometriosis (EMs) has not been entirely clarified, and different specialists hold different standpoints and different frame of reference. Sampson's theory has been comprehensively accepted and considered to be one of the main pathogenesis of the endometriosis. Furthermore, some immanent molecular-biological factors, such as the diversification of tumor gene and tumor suppressor gene, P450, unconventional expression of some cellular factors and MMP, being devoid of 17 β -HSD type Ⅱ and the counterwork of progesterone that engendered by some local tissues. In recent years, endometriosis has caused more and more attention of the clinical workers for its ascensive incidence.The tumor suppressor gene phosphatase and tension homologue deleted on chromosome 10, which is identified as PTEN gene, is found in the year of 1997. PTEN has been implicated in a large number of human tumors and is convserved from humans to worms. Characterization of PTEN protein showed that it is a phosphatase that acts on protein and on 3-phosphorylated phosphoinositides, and can therefore modulate signal transduction pathways that involve lipid secondmessengers. Recent results indicate that at least part of its role is to regulate the activity of the serine/threonine kinase AKT/PKB, FAK pathway and MAP kinase pathway, and thus thus influence cell survival signaling. It suppprts the phosphatase activity of the PTEN gene.Nowadays, more and more researches found out that, as the gatekeeper of the endometrium, PTEN gene just plays a very important role in the endometrium pathological lesions, while there is lack of studies about the corelationship between the PTEN gene and the pathogenesis of endometriosis.So with the foundation of prevenient researches, we commenced our experiments for the purpose of detecting the expression of PTEN gene in endometriosis in the level of tissue and cells and finding out the correlationship between the PTEN gene and the pathogenesis of endometriosis. By these means, maybe we can reveal the etiology of endometriosis and quest the effective diagnosis and therapy projects.Methods:1. The expression of PTEN in ectopic endometrium in 37 patients with endometriosis, 17 of which are adenomyosis patients and 20 are ovarian endometriosis patients, was analyzed by using hybridization in situ and immunohistochemsistry methods. The normal endiometrium of 33 patients with uterine myoma was studied as controls. All the results were compared with the clinical stages.2. Ectopic endometrium cells and normal endometrium cells were cultured in vitro. The quality of these cells were detected by Cytokeratin stain.3. The expression changes of PTEN gene were detected by fluorescent immunohistochemisty with the help of FITC-stained antibody.4. The different expression of PTEN proteins in ectopic endometrium cells and normal endometrium cells were also detected by flow cytometry.5. After being cultured with different does of gestrinone for different time, thecells were examined by MTT assay.6. Ectopic endometrium cells were divided into 3 groups according to the different treatments to them. Such 3 groups are control group, gestrinone group and gestrinone + PD98059 group. The proliferation changes were detected.7. Transmission electronic microscope, TUNEL assay, flow cytometry and nucleus DNA agarose gel electrophoresis to detect the apoptotic changes in ectopic endometrium cells in 3 groups.8. The changes of PTEN gene, ERK and p-ERK expressions were detected by western-blot analysis.Results:1. The level of the PTEN expression in the ectopic endometrium was decreased significantly as compared with that in the normal endometrium (P=0.0002). Of the 37 endometriosis cases, 54.1% were positive for PTEN protein and 48.6% for PTEN mRNA, all the positive signals were mainly seen in the cytoplasm.2. The expression of the gene showed a cyclic change both in patients with endometriosis and in controls. The PTEN staining was found to be more frequent in secretory phase than that in proliferative phase. But the difference was statistically meaningful only in the control group (P=0.0391, P=0.0102).3. No significant value was found in the two groups of adenomyosis and ovarian endometriosis (P=0.5915, P=0.4018). Notably, expression of the parameter has a significant correlation with the stage of the endometriosis (P=0.0021,P=0.0026).4. There is a remarkable corelationship between the expression of PTEN protein and mRNA in ectopic endometrium of endometriosisO=1.0000,P=0.0000) .5. Separate and culture the ectopic endometrial cells of 13 endometriosispatients and eutopic endometrial cells of 10 women who with the normal endometrium. All the cultured cells are provided with the hallmark of good quality.6. Detected the expression of PTEN protein by FCM and fluorescent immunohistochemisty. The difference in PTEN expression was founded between the ectopoc endomerium cells and normal endometrium cells.7. Gestrinone at different concentrations can inhibit the growth and proliferation of ectopic endometrium cells in a does-and time- dependent manner. The inhibit percentage of the cell growth after exposed to gestrinone for8, 16, 24, 32, 40 and48 his21.8%, 21.2%, 37.9%, 34.2%and33.1%, in the 10"6 mol/L group, and that is 26.9%, 42.3%, 64.2%, 70.8% and 85.2% in the 10"4 mol/L group (P<0.05), and the cell growth curve was changed accordingly.8. After 48 hours exposure of gestrinone or gestrinone and PD98059, some apoptotic changes of the cells were observed under transmission electron microscope, DNA ladder and TUNEL detection.9. FCM showed that after the expose of gestrinone or gestrinone and PD98059 group, the apoptotic rate of ectopic endometrium cells was significantly increased when compared with the control group (P<0.05). Cell cycle determined by flow cytometry showed that the percentage of Gi phase increased, but the percentage of S phase decreased; one visible apoptosis peak in front of the Gi phase was also found both in gestrinone group and gestrinone + PD98059 group.10. Western blot detection founded that the activity of PTEN protein is increased accordingly. The consistency of PTEN protein expression in control group is (1173.69+12.11). In gestrinone group, the consistency of PTEN expression is (2198.03 + 80.49) and (3201.53 ±65.00). In gestrinone and PD98059 group, the consistency of PTEN expression is (2377.62 + 124.09) and (3431.79 ±274.49).