| Objective: Epithelial ovarian cancer(EOC)is one of the three malignant tumors in gynecology.Treatment usually involves chemotherapy and surgery,current standard treatment options are complete tumor cell reduction surgery and platinum-based chemotherapy.Epidemiological survey showed that ovarian cancer is most commonly diagnosed after menopause,but there are still about 40% of young patients undergo premature treatment-induced menopause because of surgery or radiochemotherapy.The effect of treatment-induced menopause on young women is stronger than that of natural menopause,mainly related to hot flushes,sweating,vaginal atrophy,dyspareunia,insomnia,anxiety,depression,even cause osteoporosis,fracture and increase risk of cardiovascular disease,resulting in decreased quality of life of patients and non-cancer mortality.Estrogen as a part of menopausal hormone therapy(MHT),has the remarkable effect for treatment of menopausal syndrome,osteoporosis prevention and so on,especially for premenopausal ovarian cancer patients after surgery or chemotherapy,whose estrogen drops suddenly.The origen and pathogenesis of EOC have been investigated,but still poorly understood.It is generally believed that the occurrence and development of cancer may involve the activation of oncogenes,inactivation of tumor suppressor genes,apoptosis inhibition and cell cycle disorders,among which one or more genes are involved.Research of tumor growth and development caused by tumor suppressor gene mutation has been paid close attention,ARID1A,a subunit of the chromatin remodeling SWI/SNF complex,have been found to be mutated in a wide variety of tumors.And it plays an important role in the occurrence and development of ovarian clear cell carcinoma.Studies have shown that ARID1A may interact with the PI3 K pathway,which affects the phosphorylation level of Akt,so as to control the proliferation of tumor cells.Degradation of extracellular matrix(ECM)is an important step in the distant metastasis of malignant tumor cells,matrix metalloproteinase 9(MMP9)is a member of the matrix metalloproteinase family-dependent Zn2+,also known as gelatinase B,its major action on the proteoglycan types IV,X,VII,V,XI collagens and protein polysaccharide,elastin and fibronectin,is to degrade the extracellular matrix and basement membrane to achieve the role of invasion and metastasis.p53 is a tumor suppressor gene with high mutation rate in gynecological malignant tumors.Wild type p53 protein has the function of regulating cell cycle,inducing cell apoptosis,inhibiting angiogenesis and promoting DNA repair.Studies have shown that the positive rate of p53 protein in ovarian cancer tissue and adjacent normal tissue is significantly different,suggesting that the abnormal expression of p53 protein and the proliferation and the development of tumor cells has close contacts.p53 protein can regulate cell cycle,inducing cell apoptosis,inhibiting angiogenesis and promote DNA repair function.Studies have found that p53 can form complexes with ARID1A/BRG1 and transcriptional regulation of downstream genes and also with the ARID1A C-terminal interaction plays a negative role in cell cycle regulation.Some studies have shown that there is a certain relationship between the occurrence of ovarian clear cell carcinoma and the effect of estrogen,the study of estrogen induced ovarian clear cell carcinoma recurrence is rare,there is still a lot of controversy about the safety of estrogen in patients with ovarian clear cell carcinoma.By detecting the effect of different concentrations of 17 estradiol(E2)on ovarian clear cell carcinoma ES2 cells,including proliferation,invasion and ARID1A,MMP9,p53 protein expression of the cell line,we try to explore the possible effect of estrogen on ovarian clear cell carcinoma,and to provide theoretical basis for the safety of hormone therapy after the operation of ovarian clear cell carcinoma.Methods: The human ovarian clear cell carcinoma cell line ES2 were cultured in RPMI 1640 culture solution which contained 10% fetal bovine serum at 37℃,under a humidified 5% CO2 atmosphere.Cells were used in the experiment after they entered the logarithmic phase.The experiment established the blank control group(excluding E2 and solvent),the solvent control group(DMSO 0.01%v)and group E2(10-7mol/L,10-8mol/L,10-9mol/L,10-10mol/L,10-11mol/L).Cells were stripped from endogenous steroids by changing the medium to phenol red-free RPMI 1640 containing 10% charcoal-dextran stripped fetal bovine serum 24 h before treatment.All treatments were repeated for at least 3 times in each experiment.1 Cell proliferation assay(MTS method): ES2 cells treated with different concentrations of E2(10-7mol/L,10-8mol/L,10-9mol/L,10-10mol/L,10-11mol/L)for different time(24h,48 h,72h),at the same time,the blank control group and the solvent control group were set up and the differences between the two groups were observed.After the cells adherence,the time of replacement of the culture medium was considered as 0h,the cells were cultured 24 h,48h,72 h respectively in the replacement mediums,adding MTS 10 μ l each hole and cultured for 1~4h.The absorbance value of each hole was measured at 490 nm.The experiment was repeated 3 times.2 Cell migration experiments: experiment group and control group were inoculated to the Transwell cells,there were 3 holes in each group,continuous culture for 24 h,remove the Transwell chamber,fixed by methanol,crystal violet staining.Counting under the microscope(200×)the number of cells that move to the lower layer of a microporous membrane,33% acetic acid for extraction and decolorization.The absorbance was detected at 570 nm to evaluate the migration ability of tumor cells.3 Cell invasion assay: 50 mg/L Matrigel in 1:8 dilution before the experiment,using diluted coated Transwell cell polycarbonate membrane of the upper chamber,the experimental group and the control group ES2 cells were cultured in the upper chamber,with 3 wells in each group,after cultured for 24 h,fixed with methanol,crystal violet staining.Counting the number of cells in the lower layer of the membrane to assess the invasive ability of cells.4 Western blot: control group and different concentration of E2(10-7 mol/L,10-8 mol/L,10-9 mol/L,10-10 mol/L,10-11 mol/L)for different time(48h,72h),ARID1A protein,p53 protein and MMP9 protein.5 SPSS 21.0 statistical software was used for statistical analysis.The data presented as the mean±SD.Analysis among multiple groups were compared using one-way ANOVA(One-Way ANOVA).If the difference was statistically significant,the SNK method was used for multiple comparisons among groups.Differences with P﹤0.05 were considered to be statistically significant.Results:1 The effects of E2 on cells proliferation after 24 h,48h,72 h was detected by MTS,results showed that the detection of 3 time points in the same time,the solvent group compared with the blank control group,the difference was not statistically significant(P>0.05).Compared with the control group,treated with the different concentration of E2(10-7mol/L,10-8mol/L,10-9mol/L,10-10mol/L,10-11mol/L)24h,the proliferation of ES2 cell line showed increased,the difference was statistically significant(P<0.05)but no significant differences between the groups(P>0.05);treated with the different concentration of E2(10-7mol/L,10-8mol/L,10-9mol/L,10-10mol/L,10-11mol/L)48 and 72 h,the proliferation of ES2 cell line showed increased,the difference was statistically significant(P<0.05),and significant differences between the groups(P<0.05),compared with the control group,E2 is capable of promoting cellproliferation,and cell proliferation increased with the increase of E2 concentration(P<0.05).2 The results of transwell showed that compared with the control group,the absorbance value of E2 group 24 h migration cell was statistically significant(P<0.05).The number of the transmembrane cells in the E2 group was significantly more than that in the control group(73±6.2、65±4.7、61±3.5、58±1.1、58±2 vs 46±1.5),the difference was statistically significant(P<0.05).3 The results of transwell assay showed that after treatment with different concentrations of E2,the number of the transmembrane cells in the treating group was significantly more than that in the control group,the difference was statistically significant(P < 0.05).4 The western blot results showed that after 48 and 72 h since the various concentrations(25,50,and 75μmol/L)of E2 had effect on the ES2 cells,the expression level of ARID1A had significantly decreased compared with the control group and it was decreased with the increasing of the concentration of E2,then the expression level of p53 and MMP9 protein had significantly increased compared to the control group,and they were raised with the increasing of the concentration of E2.Conclusions:1 E2 could effectively enhance the ability of cell migration.2 E2 could enhance the migration ability of ES2 cells.3 E2 enhanced the invasion ability of ES2 cells.4 E2 up-regulate the expression of p53 and MMP9 protein,while down-regulate the expression of ARID1A protein.5 Estrogens should be used with caution in women after the surgery of ovarian clear cell carcinoma. |
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