The Role Of Cyclin B1/Cdc2 And Polo-like Kinase1 In The Pathogenesis Of Endometriosis

Endometriosis, the presence of viable endometrial tissue outside the uterus, affects about 10-15% of reproductive age women. This disease is frequently associated with pelvic pain and has been recognized as a major cause of infertility among women in many industrialized nations. A variety of theories have been proposed to account for this susceptibility, including genetic predisposition, aberrant immunologic response and an altered peritoneal environment and so on. But the precise pathogenesis mechanism remains largely unknown up to date. Even through endometriosis is considered a benign disorder, it exhibits some characteristics of malignancy that also are seen in malignant lesions, such as cellular atypical hyperproliferation, invasion, metastasis, neoangiogenesis and genomic instability. A relationship between endometriosis and both ovarian endometrioid carcinoma and ovarian clear cell carcinoma has been suspected for a long time. Up to 1% of women with endometriosis will develop endometriosis-associated neoplasm. Studies suggestthat the relative risk of ovarian cancer in women with a diagnosis of endometriosis is 1.9. The risk increases to 4.2 for subjects with a long history of ovarian endometriosis. Now the hypothesis is commonly accepted that endometriosis may be viewed as a neoplasm process.As it is known, hyperproliferation and malignant transformation are major characteristics of neoplasm. Early studies suggested that the proliferation was upregulated progressively during the development of endometriosis and the malignant transformation were usually found associated with endometriosis. However, little is known about the molecular mechanisms that govern cell proliferation and malignant transformation in endometriosis. Cyclin B1-Cdc2 and PLK1 are the key components of the cell cycle machinery and play important roles in the transition from G2 to M phase of the cell cycle. The Cyclin B1 can also be activated by phosphorylation of PLK1, which is highly conserved from yeasts to humans. Unscheduled up or down-regulation of these cell cycle regulatory genes during the cell cycle can help cells override the death sentence, and lead to uncontrolled cell growth and malignant transformation. Overexpression of Cyclin B1 and PLK1 had been reported in malignancies of breast, colon, esophagus, ovarian, prostate and so on.Based on the discoveries above, we suspect that abnormal cell cycle regulation might be involved in the pathogenesis of proliferation associated with endometriosis. Many cell cycle regulators in G1/S have been studied in the normal eutopic endometrium and ectopic endometrium of endometriosis and carcinoma in the past. But little is kown about the regulators controlling G2/M transition of the cell cycle in endometriosis. So in the current study, we examined the cell growth and the protein expression of Cyclin B1/Cdc2 and PLK1 in the ectopic endometria from women with endometriosis and theeutopic endometria from these with and without endometriosis by methods of RT-PCR, Western blotting, immunohistochemistry, cell cuture and flow cytometry and so on.Part I Comparation of the growth charateristics of endometrial cells from women with and without endoemtriosisObjective: To investigate the possible pathogenetic mechnism of endometriosis by comparing the growth charateristics of the ectopic and eutopic endometrial cells from endoemtriosis with that of the eutopic endometrial cells from normal women in vitro.Method: 21 ectopic and 6 eutopic endometrial cells were obtained from premenopausal women who had undergone radically hysterectomy and salpingo-oophorectomy for ovarian endometriotic cysts seperatively. The eutopic endometrial cells from 6 women who undergo hysterectomy for subserousal leiomyoma were also collected. All patients had been free of any hormonal treatments before 6 monthes of the operation, aged from 23 to 47 years old. The cells were stably cultured. The cell number and mitotic figures were counted and the cell cycle was analyzed by flow cytometry.Results: The cell number and mitotic cell number countings showed that since the second day of the cells cultured, the ectopic and eutopic endometrial cells from women with endometriosis showed faster cell growth rate than the eutopic endometrial cells from normal women (p<0.05). From the second day to the fourth day of the cells cultured, the cell number and mitotic cells of the ectopic and the eutopic endometrial cells counted every 24h were significantly different comparedwith the eutopic endometrial cells from normal women (P<0.05). The cell growth reaches a stable phase at the fifth day.Conclusion: The ectopic and eutopic endometrial cells from women with endometriosis showed significantly different cell growth when compared with the eutopic endometrial cells from normal women. These results suggested that the difference of cell growth between the ectopic and eutopic endometrial cells from women with endometriosis and the eutopic endometrial cells from women without endometriosis may be one of the basic pathogenesis mechanisms of endometriosis. Part II The expression of Cyclin B1, Cdc2 and PLK1 in endometriosisObjective: To investigate the role of Cyclin B1/Cdc2 and PLK1 in the pathogenesis of endometriosis by expressing them in the ectopic and the eutopic endometria from women with and without endometriosis.Method: RT-PCR, Western blotting and immunohistochemistry were performed to examine the expression of CylcinB1, Cdc2 and PLK1 in the 29 ectopic endometrial tissues from women with endometriosis and the eutopic endometrial samples from women with (20) and without (30) endometriosis. At the same time, Ki-67 was also expressed by immunohistochemistry staining in the three group endometrial tissues.Results: The mRNA and protein expression levels of CyclinB1 and PLK1 in the ectopic and eutopic endometrial tissues from women with endometriosis were significantly higher than that in the eutopic endometrial tissues from normal women (p<0.05) throughout the menstrual cycle. The mRNA and protein expression levels of Cyclin Bl and PLK1 at proliferative phase were higher thanthose at the secretory phase. But no significant differences of the mRNA and protein expression of Cyclin B1 and PLK1 were found between the ectopic and eutopic endometrial tissues from women with endometriosis (p>0.05). The major immunohistochemistry staining of Cyclin B1, Cdc2 and PLK1 was detected both in the nucleus and in the cytoplasm of the three group endometrial tissues. Moreover, the immuno-staining of Cyclin B1 and PLK1 in the ectopic and eutopic endometrial tissues from women with endometriosis was higher than that in the eutopic endometrial tissues from women without endometriosis. The mRNA and protein expresson of Cyclin B1 and PLK1 showed the same expression tendency. The mRNA and protein expression levels of Cdc2 remained invariable in the three group endometrial tissues throughout the menstrual cycle. The immuno-staining of Ki-67 showed that at both the proliferative and the secretory phases, the positive staining of Ki-67 in the ectopic and eutopic endometrial tissues from women with endometriosis were significantly higher than that in the eutopic endometrial tissues from normal women (P<0.05). No significant difference of the Ki-67 expression in the ectopic endometrial tissues was detected compared with the eutopic endometrial tissues from women with endometriosis (P>0.05). The staining intendency in the ectopic and eutopic endometrial tissues from women with endometriosis was higher than the eutopic endometrial tissues from normal women. The correlationship analyzed by the mothod of Pearson Correlationship showed that the immuno-staining intendency of Ki-67 in the three groups of endometrial tissues were found positively correlated with the cell growth and the protein expresson levels of Cyclin B1 or PLK1 in the three group tissues which had been detected in this study of the Part I. The correlation coefficients of between Ki-67 expression and Cyclin B1 and PLK1 protein expression levels were 0.701 and 0.752 respectively.Conclusion: The mRNA and protein expression of CyclinB1 and PLK1 in the ectopic and eutopic endometrial tissues from endometriosis were higher than those in the eutopic endometrial tissues from women without endometriosis. The correlation between Ki-67 expression and Cyclin B1 and PLK1 protein expression levels suggested that Cyclin B1 and PLK1 may be new molecules marking cellproliferation ability in endometriosis.Part III The effect of Alsterpaullone on cell growth of endometriosisObjective: To investigate the role of Cyclin B1/Cdc2 in the mechanism of pathogenesis of endometriosis.Mehtods: The ectopic and eutopic endometrial cells from endoemtriosis and the eutopic endometrial cells from normal women were collected and stably cultured as described in this study of the Part I. Before and after the cells treated with Alsterpaullone (Alp), the mitotic cell number was counted and the cell cycle was analyzed by flow cytometry. At the same time, the protein expression of Cyclin B1 and PLK1 were analyzed by Western blot before and after the cells treated withAlp.Results: When the cells were treated with Alp at a concentration of 0-3 mM/L for1h, the inhibiton effects of Alp on the three groups of the endometrial cells all showed no significant difference compred with those of their control cells (P>0.05). When the cells were treated with Alp at a concentration of 5 mM/L and 10 mM/L for 1h, the three groups of the endometrial cells were observed significantly inhibited by Alp compared with their control cells(P<0.05). Afetr the cells treatedwith Alp at a concentration of 5 mM/L for 24h, both the cell growth and the Cyclin B1 and PLK1 protein expression showed significant reduced by Alp in the all three group cells when compared with their control cells (P<0.05). After the cells treated with Alp for 48h and 72h, the cell growth and the protein expression of Cyclin B1 and PLK1 in the ectopic and the eutopic endometrial cells from women with endometriosis were observed both significantly inhibited compared with their control cells (P<0.05). But the cell growth and the protein expression of Cyclin B1 and PLK1 in the eutopic endometrial cells from normal women were not observed significantly inhibited compared with their control cells (P>0.05). There was no significant difference of cell growth and protein expression of Cyclin B1 and PLK1 between the ectopic and the eutopic endometrial cells from the same endometriosis after the cells treated with Alp for 24h, 48h and 72h (p>0.05).Conclusion: Cyclin Bl/Cdc2 may be involved in the higher cell growth ofendometriosis and thus mediates the pathogenesis of endometriosis.It may be usedas a molecular target in the treatment of endometriosis in the future.Part IV The effect of siRNA targeting human PLK1 on cell growth of endometriosisObjective: To further investigate the role of PLK1 in the pathogenctic mechanisms of endometriosis.Methods: The ectopic and eutopic endometrial cells from endoemtriosis and the eutopic endometrial cells from normal women were collected and stably cultured as described in this study of the Part I. Before and after the cells infected with siRNA targeting human PLK1, the cell cycle was analyzed by flow cytometry andthe protein expression of Cyclin B1 and PLK1 were analyzed by Western blotting before and after the cells infected with si RNA targeting human PLK1. Results: Afetr the cells infected with siRNA targeting PLK1 for 48h, the ectopic and eutopic endometrial cells from women with endometriosis showed significantly retarded cell growth and reduced protein expression of Cyclin B1 and PLK1 compared with the control cells (p<0.05). The results analyzed by flow cytometer showed that the cell number in G2/M phase were significantly increased by 79.15% and 71.21% (p<0.05) in the ectopic and the eutopic endometrial cells respectively when compared with their control cells.But it was just increased by 26.23% (P>0.05) in the eutopic endometrial cells from normal women compared with their control cells. The analyses from Western blotting showed that the protein expression of Cyclin B1 and PLK1 were significantly inhibited in the ectopic (p<0.05) and the eutopic (p<0.05) endometrial cells from women with endometriosis after the cells treated with siRNA for 48h compared with their control cells, but not significantly inhibited in the eutopic endometrial cells from normal women (p>0.05).Conclusion: PLK1 may be elimilated partly by the molecule of siRNA targeting human PLK1 in the ectopic and eutopic endometrial cells from women with endometriosis. So the siRNA targeting human PLK1 may be used as one of potential methods for the therapy of endometriosis in the future.