DNA hypomethylation of the COX-2 gene promoter is associated with up-regulation of its mRNA expression in eutopic endometrium of endometriosis

DanBo Wang 0 1 Qi Chen 1 ChiYuan Zhang 1 Fang Ren 1 Tong Li 1 0 Department of Obstetrics & Gynecology, Shengjing Hospital Affiliated to China Medical University , 36 Sanhao Street, Shenyang 110004 , People's Republic of China 1 Department of Obstetrics & Gynecology, Shengjing Hospital Affiliated to China Medical University , Shenyang 110004 , People's Republic of China Background: Accumulated evidence reveals that cyclooxygenase-2 (COX-2) was overexpressed in eutopic endometrium of endometriosis, which may play a critical role in the pathogenesis of endometriosis. However, few studies have been performed to explore the molecular mechanisms underlying the abnormal high expression of COX-2 in endometriosis. Considering the fact that a number of recent studies have shown DNA methylation affecting some genes in endometriosis, the present study was therefore aimed to determine whether the observed high expression COX-2 in endometriosis is caused by the hypomethylation of CpG island within the promoter of this gene. Methods: The endometrial tissues were collected from 60 women with endometriosis (endometriosis group) and 20 women without endometriosis (control group). The methylation status of COX-2 was examined by methylation specific PCR. Quantitative real-time RT-PCR was performed to measure COX-2 mRNA level in endometrial tissues. Results: The frequency of promoter hypermethylation of COX-2 was lower in eutopic endometrium of the endometriosis group (41.7%) than that in the control group (75.0%), P < 0.05. COX-2 mRNA level in the eutopic endometrium of the endometriosis group was 2.61-fold higher than that in the control group (P < 0.01). COX-2 mRNA level in unmethylated endometrium of the endometriosis group or the control group was 2.39-fold and 2.66-fold, respectively, higher than that in the methylated endometrium of the same group (P < 0.01). Conclusions: The hypomethylation within the promoter of COX-2 may be responsible for the elevated gene expression in eutopic endometrium of endometriosis. - Background Endometriosis is an estrogen-dependent gynecological disorder that affects 6-10% of women of reproductive age. It is characterized histologically by the presence of endometrial tissue at sites outside of the uterine cavity, primarily on the pelvic peritoneum and ovaries, resulting in severe pelvic pain, pain during intercourse, and infertility [1,2]. To date, the etiology and pathogenesis of endometriosis remain largely unknown. Endometriosis is a benign gynecological disease with malignant behaviors, such as enhanced proliferation and cell invasion, ectopic implantation of distant organs similar to the tumor metastasis. The eutopic endometrium of patients with endometriosis has various alterations compared with endometrium of healthy women [3]. Aberrant expression of genes in eutopic endometrium was reported be involved in cell adhesion, invasion, and angiogenesis, therefore it was quite critical to the pathogenesis of endometriosis [4-6]. The ectopic endometrium of endometriosis often behaves unpredictably; it can vary from microscopic foci to large, grossly visible, endometriotic cysts, which leads to difficulties in research, diagnosis, and treatment. Eutopic endometrium of endometriosis is readily available and gene alteration in the eutopic endometrium can be easily detected. Identification endometriosis-related genes in eutopic endometrium will further reveal the pathogenesis of endometriosis and offer the basis for targeted gene diagnosis and therapy of endometriosis. In the previous study, we identified 10 up-regulated genes in the eutopic endometrium of endometriosis during the secretory phase using cDNA-RDA and found that cyclooxygenase-2 (COX-2) was one of the up-regulated genes [7]. As the key enzyme in the conversion of arachidonic acid to prostaglandins (PGs), COX-2 can be induced by growth factors, oncogenes, and tumor promoters, and has been mainly associated with the inflammatory response [8]. Its elevated expression in the eutopic endometrium has also been reported to be associated with endometriosis [9,10]. However, the underlying mechanism of overexpression of COX-2 in eutopic endometrium of endometriosis has not been well defined. DNA methylation is an epigenetic phenomenon known to play a critical role in the regulation of gene expression in development, differentiation, and complex diseases, with cancer being the most prominent example [11,12]. Moreover, aberrant methylation of promoter CpG island of the COX-2 has been known as an alternative mechanism of its abnormal expression and contributes to the carcinogenesis in many human cancers [13,14]. Recently, DNA methylation has also been shown to affect a number of genes in endometriosis [15]. These findings lead us to investigate whether aberrant expression of the COX-2 in eutopic endometrium of endometriosis is caused by aberrant methylation of the COX-2 CpG island. The nuclear factor responsible for the interleukin-6 expression (NF-IL6) site as one of the critical cites of the COX-2 promoter plays an important role in the regulation of COX-2 expression [16]. In the present study, we investigated, for the first time, that whether the observed elevated expression of the COX-2 gene in endometriosis is associated with the hypomethylation of NF-IL6 site within the promoter of this gene. Methods Patients and specimens Eutopic endometrium samples were collected from 60 patients with an average age of 43.65 3.99 years, who underwent hysterectomy due to endometriosis stages III and IV according to the Revised American Fertility Society Classification for Endometriosis at the Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University. For controls, endometrium samples were obtained from 20 women with an average age of 43.20 2.87 years, who underwent total hysterectomy due to cervical intraepithelial neoplasia III in the same hospital, surgically confirming without endometriosis. Diagnosis was confirmed with histopathological examination in all cases. All subjects presented regular menstrual cycles (cycle length was approximately 25 to 32 days). Cycle stage was estimated according to the date of the last menstrual phase or by histological evaluation [17]. All specimens were obtained in the secretory phase of the menstrual cycle (days 15 to 28) in our study. As shown in Table 1, there was no difference between the two groups with respect to the cycle phase. None of these patients had received any GnRH analogue, antibiotics, radio-, chemo-, or hormone therapy in the last 6 months prior to the surgery. Endometrium samples were gathered within 1015 min after hysterectomy and immediately frozen in liquid nitrogen and then preserved in 80C refrigerator until further use. Written informed consent was obtained before surgical procedures, including a consent form and protocol approved by the Institutional Review Boards of China Medical University. Methylation specific (...truncated)