The Correlation Between AT1R And MAPK Signal Transduction Pathway On Estrogen Induced Ishikawa

Endometrial carcinoma is now the most common pelvic genital cancer in women.It is about 7% all female carcinoma,endometrial carcinoma threaten women healthy acutely,and its mechanism is rather complex.High level estrogen stimulus long-term is most closely with endometrial cancer, AT1R is hyper-express in many tumor cells. In order to investigate estrogen induced ishikawa wether through AT1R activate endometrial cancer MAPK signal transduction pathway.Our study apply immunofluorescence to ivsestigate the expression of AT1R, Cell proliferation was detected using MTT,and cell cycle and apoptosis were determined using flow cytometry on estrogen induced and saralassin effect of ishikawa. We targeting AT1-R mRNA,then,cell proliferation was detected using MTT assay, and cell cycle and apoptosis were determined using flow cytometry, and the expression of ERK1/2 by western bloting.Materials and Methods1. Materials(1) cell line the ishikawa cell line of endometrial cancer is origin of O&D laboratory of the university of Japan. estrogen receptor and progesterone receptor are all masculine.(2) Reagent 17β-estrogen,saralasin, MTT, PI and AT1R siRNA.2. MethodsThrough the method of MTT we measure the best estrogen induced concentration and the most depression concentration of saralasin. Affter that, Cell proliferation was detected using MTT,and cell cycle and apoptosis were determined using flow cytometry on estrogen induced and saralassin effect of ishikawa.We targeting AT1-R mRNA,then,cell proliferation was detected using MTT assay, and cell cycle and apoptosis were determined using flow cytometry, and the expression of ERK1/2 by western bloting.3. statistical analysisSPSS 13.0 software was employed to analyze all data. Statistical evaluation was performed using One Way ANOVA, p<0.05 was considered as statistical significance.Results1. Immunofluorescence assay confirmed the expression of ATIR in ishikawa cell membrane and cytoplasm.2. The concentration of estrogen in the 10-8 mol/L on cell proliferation is the most obvious, saralasin at 10-5 mol/L to suppress ishikawa cells is the most obvious, with the above-mentioned concentrations of acting on ishikawa cells, in the 10min group of the most significant inhibition of cell, p<0.05. Therefore, we follow-up experiments using 10min, to study the cell cycle and apoptosis.3. G0/G1 cell-cycle view to reducing the estrogen group, S phase increased; saralasin group increased G0/G1 phase, S phase reduction.4. Apoptosis in the normal control group, early apoptosis rate of 9.54%, estrogen group of cells in early apoptosis rate of 6.87%, saralasin group of cells in early apoptosis rate was 13.19%, the difference between the groups was statistically significant (P< 0.01); late apoptosis and necrosis rate of 4.13% in normal control group, estrogen group was 4.79%, saralasin group was 8.54%, the difference between the groups was statistically significant (P<0.05).5. Fluorescently-labeled siRNA (GFP-siRNA) transfection resulted in the adoption of transfected cells in fluorescence intensity observed by fluorescence microscopy filter out a high transfection rate of a chain, the use of this test after transfection, AT1-R protein bands In the 24h,48h and 72h the inhibition rates were 32.82%,52.47% and 82.40%, in the next experiment, we use 72h adding processing elements.6. AT1-R siRNA on estrogen-induced cell proliferation ishikawa of siRNA-ATIR group was 87.23%, Estrogen group was 89.55%, from the relative proliferation rate of siRNA-ATIR group showed that cell proliferation after transfection, compared with the control group, female 10min of their hormone-induced proliferation was not obvious.7. Ishikawa flow cytometry cell cycle and apoptosis rate of 1.73% of natural apoptosis in transfected AT1-R siRNA-3 72h, the apoptosis rate of 14.39% of transfected AT1-R siRNA-3 72h and then by the 10-8mol/1 estrogen treatment 10min after the apoptotic rate was 13.45%. After the effects of siRNA cells in G0/G1 phase compared with the control group the ratio increased, S proportion reduced, G2/M phase changed little; estrogen group and the transfected group than in, G1/GO phase increased, S phase reduced, G2/M period of little change.8. Western blot normal ishikawa cells transfected with siRNA-ATIR and transfection of siRNA-ATIR in ishikawa cells in estrogen-treated cells, western blot detection of ERK1/2 protein expression changes, in which transfection of siRNA-AT1R group of ERK1/2 expression in decreased significantly.Conclusion1. Within two hours of estrogen stimulates the proliferation of ishikawa role in 30min in the strongest decline in the cell G1 phase, S phase increased;2. Saralasin on estrogen-induced cell proliferation of ishikawa more obvious inhibition, from the relative inhibitory rate to see, in the 10min when a relatively significantly inhibited; saralasin can increase in late apoptosis and early apoptosis.3. Ishikawa silence in the use of siRNA technology, AT1R, then estrogen for induction, and further confirmed the inhibit AT1-R gene expression in ishikawa cell proliferation was inhibited AT1-R siRNA transfection after ishikawa cell apoptosis was significantly the promotion of estrogen-induced transfected cells can promote cell proliferation; cell cycle analysis showed that after transfection ishikawa cells increased G0/G1 phase, S phase reduced, G2/M phase did not change significantly.4. Ishikawa in AT1R silence after its ERK1/2 expression decreased, indicating possible in ishikawa cells by AT1-R is the MAPK signaling pathway to regulate proliferation and apoptosis.