Study On The Relationship Between COX-2and Calcium Channel Subtypes Associate With The Pathogenesis Of Uterine Fibroids

Research BackgroundUterine fibroids is the most common benign tumor of the reproductive organ in women of childbearing age, its incidence rate has been rising year by year, the clinical incidence rate was20%-40%. The pathogenesis of uterine fibroids is still unknown. The pathogenesis of uterine fibroids including:uterine fibroids are the steroid hormone-dependent tumor, myoma of uterus is related to heredity, mutation, and so on. Abnormal expression of genes was involved in cell signal transduction, ion channel transport protein. Endometrial tissue in the uterine smooth muscle supports a wealth of prostaglandin synthesis and secretion, through autocrine and paracrine mechanisms which acts on the smooth muscle cells, involved in pregnancy and non-pregnant uterus physiological effects. We have paid our little attention to the role of the prostaglandin system in the pathogenesis of uterine fibroids.Prostaglandin (PG), the derivative of arachidonic acid (aa), is a biologically active substance. Prostaglandins are important hormones regulating uterine contractions, while cyclooxygenase (COX-2) is rate-limiting enzyme in the process of catalytizing aa into PG, and is the target of non-steroidal anti-inflammatory drugs (NSAIDs), which not only regulates the synthesis of prostaglandins, but also participates in the development and progression of tumors.COX-2is an isoenzyme COX in normal human body, which can not be detected in most of organizations. Under the induction of various cytokines, growth factors and oncogenes, the activity of COX-2is increased. Compared with normal tissue, expressions of COX-2in tumor tissue were significantly increased, inhibition of COX-2activity was directly related to the anti-tumor effect, while COX-2selective inhibitors have anti-proliferative effect. Studies have shown that, expressions of COX-2in a variety of malignancies, such as cervical cancer, ovarian cancer, colon cancer, were higher. COX-2is an immediate early response gene, COX-2is an isoenzyme of the COX, usually in most of the tissues and cells is not expressed, but were highly induced in the inflammatory reaction, hormone and tumor promoters, and there is no obvious change of COX-1content. COX-2is highly expressed in tumor tissues, is directly related to the anti tumor effect of inhibiting the activity of COX-2, and anti COX-2selective inhibitors have anti proliferation effect. COX-2derived prostaglandin E2(PGE2) is a kind of biological activity of lipid, is the major prostaglandin. Study on direct proof of PGE2promote tumor growth. Research shows that, in a variety of human malignant tumors such as breast cancer, colon cancer, prostate cancer, lung cancer, liver cancer, head and neck tumors, COX-2and PGE2expression was up-regulated. In view of the role of COX-2and PGE2in the occurrence and development of tumor, but now the expression and itscorrelation with cell proliferation of COX-2in uterine fibroids is not very clear.Previous studies showed that uterine fibroids was associated with the proliferation of estrogen, androgen and abnormal cells, but the exact pathophysiology is not known. Some studies observed uterine abnormal contraction in some patients with uterine fibroids. Recent studies have shown that, uterine deep tissue, i.e. subendocardial muscle tissue, appears "creep shrinkage", and smooth muscle cells function declines but enhances diffusion. All these studies indicate that abnormal smooth muscle contraction may play an important role in the pathophysiological process of uterine fibroids, but its potential molecular mechanism is still unclear, and need further research. Experiments showed that expression of COX-2and secretion of PGE2in fibroblasts is regulated by intracellular calcium ion concentration. Calcium ions are important upstream signaling molecules initiating cell contraction. As is known, uterine smooth muscle excitation-contraction coupling linking is regulated by double signal transduction pathways, in which calcium ion signal transduction pathway plays an important role in cellular stress process. Calcium channel proteins is a calcium channel signal transduction molecules switch, they regulate not only cell contraction but also the cell cycle, which is closely associated with many tumors and carcinogenic path. Transient calcium channel (TRPC) is a transmembrane calcium channel protein mediating cell Ca2+influx, the regulation of cell contraction and cell cycle, and is closely related to the development and occurrence of multiple tumors. In recent years, researches in the field of calcium channel protein have made significant achievements.According to reports, TRPC is associated with abnormal uterine contraction, though it is not clear that if it is related with uterine myoma proliferation.Currently, people’s understanding of prostaglandins and calcium channel in uterinefibroid tissue is very superficial, we need further confirmation about some important aspects. In this study, we observed COX-2expression in the uterine fibroid tissue and normal muscle tissue, and its correlation to the proliferation of cells of uterine fibroid in in order to identify the correlation between COX-2and the incidence of uterine fibroid. We also studied the expression of calcium channel protein in uterine fibroids cells, and its effect on fibroid cell proliferation and PGE2. The findings may have important significance to further strengthen our understaning about the mechanisms of the development of uterine fibroids, and to prevent and treat the disease. PART I STUDY OF EXPRESSIONS OF CYCLOOXYGENASE-2AND PGE2SECRETION IN UTERINE FIBROIDSObjective:To investigate the expression of cyclooxygenase-2(COX-2) and secretion of PGE2in uterine fibroids.Methods:(1) We collected uterine fibroid tissues and their paired adjacent healthy uterine smooth muscle tissues from30cases of uterine fibroid cases. Cultured human uterine fibroids smooth muscle cell separation and uterine smooth muscle cells.(2) We used immunohistochemistry and quantitative real-time PCR, as well as western blot to detect COX-2expression.(3) Immunofluorescence detection of expression of COX-2in smooth muscle cells and uterines fibroids cells.(4) MTT method was used to detect the proliferation assay.(5) ELISA experiment:ELISA kit for detection of normal smooth muscle cells and secretion in cultured fibroids cells24hours after PGE2amount.Results:(1)We have mastered the uterine fibroids cells method for in vitro culture, to establish an isolation and clinical samples of fibroids and normal smooth muscle cells cultured in vitro.(2)COX-2was detected by immunohistochemistry in both uterine fibroids and uterine smooth muscle, the positive index of smooth muscle cells was11.90and the positive index of uterine fibroids cells was46.50(*P<0.05). The western blot results showed that COX-2expression was significantly higher in uterine fibroid cases(0.872±0.035), as compared to the expression in urerine smooth muscles(0.202±0.056). Fluorescence quantitative PCR showed that the expression of COX-2mRNA in uterine fibroids was higher than that of normal smooth muscle tissue,*P<0.05.(3)Proliferation rate of uterine fibroids was about1.48±0.09times than normal smooth muscle cells,*P<0.05. (4)Adding10μM of NS-398or celecoxib into cultured uterine fibroids cells and normal smooth muscle cells respectively, after incubation for24hours, add MTT to assay cell proliferation, we found the proliferation of cells from uterine fibroids was significantly inhibited, but normal uterine smooth muscle cells proliferation was not significantly affected.(5) Secretion of PGE2of uterine fibroids was significantly higher than that of normal smooth muscle cells,*P<0.05.(6) Adding10μM of NS-398or celecoxib into cultured the smooth muscle cells of uterine fibroids and normal smooth muscle cells respectively, after incubation for24hours, add MTT to assay cell proliferation, we found the secretion of PGE2of uterine fibroids was significantly inhibited, but normal uterine smooth muscle cells proliferation was not significantly affected.Conclusion:COX-2expression in uterine fibroids was significantly higher, the secretion of PGE2of uterine fibroids was significantly inceased, the proliferation of cells from uterine fibroids was significantly improved, compared to the control group, which are all inhibited by NS-398or celecoxib, suggesting that COX-2plays an important role in the pathogenesis of uterine fibroids. PART Ⅱ STUDIES OF EXPRESSION OF CALCIUM CHANNEL SUBTYPES AND THEIR RELATIONS WITH PGE2SECRETION INDUCED BY COX-2IN UTERINE FIBROID OF PATIENTSObjective:To investigate expression of calcium channel subtypes and their relations with PGE2secretion induced by COX-2in uterine fibroid of patients. Use GIPZ lentiviral vector carrying TRPC1shRNA or TRPM7shRNA to perform RNA interference experiments. We observe the changes of uterine leiomyoma cell proliferation, PGE2, and calcium secretion.In order to understand the relationship of calcium signal transduction pathway between the uterine fibroids.Methods:(1)We collected uterine fibroid tissues and their paired adjacent healthy uterine smooth muscle tissues from30cases of uterine fibroid cases. Cultured human uterine fibroids smooth muscle cell separation and uterine smooth muscle cells.(2)Use Laser scanning confocal microscopy to detect intracellular free calcium concentration.(3) We used quantitative real-time PCR, as well as western blot to detect expression of calcium channel subtypes.(4) Use GIPZ lentiviral vector carrying TRPC1shRNA or TRPM7shRNA to perform RNA interference experiments.(5)MTT method was used to detect the uterine leiomyoma cells proliferation after perform RNA interference of TRPC1and TRPM7.(6)ELISA kit for detection PGE2secretion of uterine leiomyoma cells after RNA interference of TRPC1and TRPM7.(7) Use Laser scanning confocal microscopy to detect calcium concentration after RNA interference of TRPC1and TRPM7. Results:(1)The laser confocal microscope to detect intracellular free calcium concentration showed calcium concentration in fibroids group (25.443±3.203, n=30) was significantly higher than that in normal muscle group (13.223±1.450n=30),*P <0.05.(2)PCR analysis showed, expressions of TRPM7and TRPC1in uterine fibroids were significantly higher than those in normal smooth muscle tumors (*P<0.05), while there was no difference on the other calcium channel subtypes between the two groups.The western blot showed, expressions of TRPM7and TRPC1in uterine fibroids were significantly higher than those in normal smooth muscle tumors (*P <0.05), and there was no difference on the other calcium channel subtypes between the two groups too.(3)After using shRNA virus leiomyoma significantly reduced TRPC1expressions of cells, we found cell proliferation of uterine fibroids was significantly inhibited and secretion of PGE2was significantly inhibited,calcium level decreased.(4) After using shRNA virus leiomyoma significantly reduced TRPM7expressions of cells, we found cell proliferation of uterine fibroids was significantly inhibited and secretion of PGE2was significantly inhibited,calcium level decreased.Conclusion:Calcium channel subtypes TRPC1and TRPM7exhibit different expression patterns in uterine fibroids and surrounding smooth muscles, suggesting that calcium signaling pathway regulated by these calcium channel proteins may be associated with the incidence of uterine fibroids. CREATIVE POINTS IN THIS STUDY1.We researched the role of COX-2and PGE2in the development of uterine fibroids, in order to provide new ideas for studying the mechanism of pathogenesis of uterine fibroids.2. We studied NSAIDs on the development of uterine fibroids, in order to provide new ideas for the prevention and treatment of uterine fibroids.3. We found a significantly higher expression of TRPC1and TRPM7calcium channels in uterine leiomyoma cells, and through RNA interference silencing of both proteins, we found the proliferation of leiomyoma cells was significantly inhibited, and the PGE2secretion was significantly inhibited, indicating that TRPM7and TRPC1may be involved in the development of uterine fibroids, and there might be crosstalk between between the path of calcium ion transport mediated by TRPC1and TRPM7and PGE2secretory pathway induced by COX-2.