Screening And Identification Of Differentially Expressed Genes In Endometriosis Using CDNA Microarray

Background:Endometriosis is common gynecological disease. EMS is characterized by the presence of abnormally located tissue resembling the endometrium with glands and stroma. The etiology of EMS is still unknown. We hope explain the cause of EMS at molecule level, search for the molecule markers and explore the gene therapy of EMS, and fully investigate the differentially expressed genes between eutopic and ectopic tissues. Screening related genes is the bases and directions to improve the level of disease prevention and cure. Wholly understanding of the changed gene expression patterns between eutopic and ectopic tissues, and screening the related genes would provide a credible key to solve these problems. DNA microarray, which combined molecular biology and microelectronics, is the new DNA analysis and examining technology. DNA microarray has been widely applied in the gene expression now.Part I Screening differentially expressed genes in EMS bycDNA microarrayObjective: To screen the differentially expressed genes between eutopic andectopic tissue using cDNA microarray and analyse the function of these genes initially. Methods: Patients who were scheduled for surgery for EMS (n =6) were recruited to participate in this study, and their informed consent was obtained. None of the patients were on hormonal therapy before the surgery. All six patients were in the follicular phase of the menstrual cycle at the time of the surgical procedure. Samples of EMS tissue were obtained from ectopic sites in the abdomen. In each patient, the cyst wall of the endometrioma was removed during laparoscopy. The cyst wall was irrigated with PBS and divided into two half. One half was immediately placed in liquid nitrogen for transport to the laboratory. The other half was sent to the pathology laboratory. Histopathology confirmed that the cyst walls were endometriomas. Eutopic endometrial tissue was obtained from the uterus of the same patients at the time of the laparoscopy by performing hysteroscopy, followed by dilatation and curettage. The total RNA was extracted from tissues. Both kinds of RNA from study group (ectopic tissues) and control group (eutopic tissues) were reversal transcribed to the cDNA with the incorporation of fluorescent to prepare the hybridization probes. The mixed probes were hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for the fluorescent signal and showed differences between study group and control group. Results: Among the 578 target genes, 9 genes differentially expressed prominently in all 6 samples were identified, 2 genes were up-regulated and 7 genes down-regulated. The 9 genes were IL-12RB1, MEMD, ITGA6, CD9, CD73, RAB2, TNFSF13, ICAM-1 and ARHI.Conclusion: cDNA microarray technique is effective in screening the differentially expressed genes between eutopic and ectopic tissue.Part II Identification of differentially expressed genes in EMSObjective: To identify of the two differentially expressed genes, ICAM-1 and ARHI.To investigate the relationship between ICAM-1 and ARHI levels and the pathogenesis and development of EMS. Methods:1. Endometrial samples were collected from eutpoic tissues (n=18), ectopic tissues (n=23) and endometrial tissues without EMS (n=17) .All samples were immediately placed in liquid nitrogen for extracting RNA and protein. Eutopic endometrial tissue with EMS was group A, ectopic endometrial tissue with EMS was group B, Eutopic endometrial tissue without EMS was group C.2. The level of 1CAM-1 mRNA in tissues were detected by Quantikine mRNA Kit.3. We designed ICAM-1 gene specific primer by Primer Primer 5.0 software. The sense primer sequence was 5'CAAGGTGACGCTGAATGG 3'. The anti-sense primer primer sequence was 5'TTATGACTGCGGCTGCTAC 3'.PCR product was 464bp.TANON image analysis system calculated the intensity in each group and analyzed the level of ICAM-1.4. The specimens were analyzed by ICAM-1 ISH kit (group A, n=15; group B, n=18;group C, n=15). Specific probe sequences are:a: 5' -...