Expression Of Cyclina And P21^WAF1/CIP1 In Ectopic And Eutopic Endometrium Tissues And Their Clinical Significance

Endometriosis is a common disease in women of reproductive years. The common symptoms are dysmenorrhea,chronic pelvic pain, intercourse discomfort and infertility, have serious effect on the healthiness and life quality to these women. The incidence of this disease is moving up gradually, the incidence is about 7%-15%,in the women of infertility is about30%-50%.The Pathogenesis is not very clear now.A variety of the theories have been proposed to account for it,including endometrial implantation theory, metaplastic theory of the body cavity epithelium, induction theory.Even through endometriosis is considered a benign disorder,it exhibits some characteristics of malignancy tumor,such as cell proliferation, invasion, metastasis and recurrence.A similarity between endometriosis and cancer from the molecular level had been reported. As known,hyperproliferation and malignant transformation are major characteristics of neoplasm, the abnormal cell division (the cell cycle) result in cell proliferation and malignant transformation. Cyclin A is an important regulatory factors in G1/S and G2/M phase,controling DNA replication, and cell mitosis, playing an important role in tumor cell proliferation and malignant transformation. P21WAF1/CIP1 (P21) is a broad negative regulatory factor, can be combined with CyclinA-CDK2 to diminish the activity of the cyclinA-CDK2 kinase,so as to inhibit cell proliferation. Now in a variety of tumor tissues have found abnormal expression of P21 and cyclinA, the high expression of cyclinA and low expression of p21 in tumor tissue are closely related with tumor cell proliferation . In recent years, researchers started to study etiology and pathogenesis of endometriosis from the perspective of tumor. Some researchers have preliminary studies on Gl/S and G2/M2 transforming in endometriosis, such as cyclinD1, B, etc.But in G1/S and G2/M phase have little observation. Zhang H, etc in china ,using proteomics technologies in patients with endometriosis,founding the high expression proteins point Seeming cyclinA, thus we speculated that the pathogenesis of endometriosis may be related to abnormal expression of p21 and cyclinA and lead to abnormal cell growth and proliferation activity changed.ObjetiveThis issue selected cyclinA and p21 as detection indexes, to investigate the expression differences in endometriotic group, eutopic endometrium group and control group,so as to investigate the possible roles of cyclinA and P21 in the pathogenesis and development of endometriosis and their clinical significance. It may be reveal the cause of endometriosis, pathogenesis and explore new treatment method and provide a new theory basis.Methods1.From May 2008 to January 2009, randomly selected open or laparoscopic surgery patients with endometriosis in the hospital of first Affiliated Hospital of Chongqing Medical University, twenty-five specimens of eutopic endometrium(eutopic endometrium group) and twenty-eight specimens of ectopic endometrium(ectopic endometrium group) were obtained from 28 patients with endometriosis.Thirty–five specimens of endometrium were also obtained from women with hydrosalpinx, tubal adhesions (control group).2.SABC immunohistochemical method was used to determine the positive rat and positive position of cyclinA and p21 protein in endometrial epithelial cells and interstitial cells .3.Western-blotting was used to detect the cyclinA and p21mRNA expreesion in endometriotic group, eutopic endometrium group and control group and their clinical significance.4.RT-PCR was used to detect the cyclinA and p21mRNA expreesion in endometriotic group, eutopic endometrium group and control group and their clinical significance. Results1.The immunohistochemical results show cyclinA protein expression was detected mainly in the nucleus of endometrial glandular cells and stromal cells, the cyclinA protein positive rate (82.1%) were significantly higher than in eutopic group (36.0%) and control group (14.3%) in ectopic endometrium (P<0.05). The immunohistochemical result show P21 protein expression was detected mainly in the nucleus of endometrial glandular cells and stromal cells, the positive rate in control group (47.6%) and in the eutopic endometrium (23.1%) were significantly higher than the ectopic endometrium group (10.7%) (P <0.05).2.The western-blotting results show the expression level of cyclinA protein in ectopic endometrium were 0.95±0.10 at the proliferative phase and 0.91±0.18 at the secretory phase , significantly higher than that in the eutopic endometrium tissues(0.53±0.08 ,0.27±0.11)and control group(0.47±0.11,0.22±0.09)(both P<0.05), but the expression level of cyclinA protein in eutopic endometrium compared with the control group ,there was no significant difference (P>0.05).The expression level of P21 protein in control group were 0.74±0.13 at the proliferative phase and 1.23±0.15 at the secretory phase ,significantly higher than that in the eutopic endometrium tissues ( 0.59±0.21,0.62±0.29 ) and in the ectopic endometrium(0.40±0.36 ,0.43±0.33) (both P<0.05).3.The expression level of cyclinA mRNA in ectopic endometrium were 1.24±0.10 at the proliferative phase and 1.33±0.21 at the secretory phase, significantly higher than that in the eutopic endometrium tissues(0.72±0.26 ,0.42±0.22)and control group(0.68±0.09 , 0.35±0.06) (both P<0.05), but the expression level of cyclinAmRNA in eutopic endometrium compared with the control group ,there was no significant difference (P>0.05).The expression level of P21 mRNA in control group were 0.21±0.01 at the proliferative phase and 0.54±0.35 at the secretory phase, significantly higher than that in the eutopic endometrium tissues(0.09±0.06 , 0.28±0.02)and in the ectopic endometrium(0.06±0.01 ,0.13±0.00) (both P<0.05).4.The expression level of cyclinA in proliferative phase of the control group was significantly higher than secretory phase. The expression level of P21 in secretory phase of the control group was significantly higher than proliferative phase. But the expression level of cyclinA and p21 in the proliferative phase and Secretory pase of the ectopic endometrium have no significant difference(P>0.05),5.It showed that cyclinA and p21 were negatively spearman correlation in the ectopic endometrium.(r=–0.738,P<0.01).Conclusion1. CyclinA and p21 are important cell cycle regulatory protein, comparing with control group and the eutopic endometrium group, cyclinA protein and gene expression in ectopic endometrium were significantly higher than in eutopic and control Group. p21 protein and gene expression in ectopic endometrium was lower than in the control group and eutopic endometrium group. This study suggests that abnormal expression of p21 and cyclinA in ectopic endometrium may play an important role in cell proliferation, which may be due to cyclinA combine with cyclinA-CDK2 as complexes to promote cells from S phase to G2 phase and G2 phase to M phase. low expression of p21 reduces inhibiting the activity of the cyclinA-CDK2 kinase,so as to let the cells continue proliferation. The findings of our research in cell proliferation aspects of endometriosis provides a new theory basis for our future research areas about the molecular target treatment for endometriosis .2. CyclinA and p21 expression in ectopic endometrium group was a negative correlation, indicating that a single high expression of cyclinA or low expression of p21 may not lead to the occurrence of endometriosis, which requires coordination between the two or other more factors.3. The expression level of cyclinA in proliferative phase of the control group was significantly higher than secretory phase. The expression level of p21 in secretory phase of the control group was significantly higher than proliferative phase, but the expression level of cyclinA and p21 in proliferative phase and Secretory pase of the ectopic endometrium have no significant difference.CyclinA was keeping high expression, but p21 keeping low expression. We speculated that cyclinA and p21 in the regulation of endometrial growth may rely on the role of estrogen and progesterone. This result further confirmed that the endometriosis is a hormone-dependent diseases from proliferation pathway, it provides a new theory basis and the target of endocrine therapy of endometriosis .