SIRT1 Expression In Endometrioid Carcinoma And The Influence And Mechanism Of SIRT1 On The Biological Behavior Of Endometrial Cancer Cells

BackgroundEndometrial carcinoma (EC)is one of the most common malignant tumors of female genital tract, which primarily origins from endometrial epithelial tissues and of which, endometrial adenocarcinoma is the most common pathological type. Its morbidity is after that of cervical cancer, but even more than that of cervical cancer in the developed countries and Beijing, Shanghai and other more developed regions in china, furthermore, there is increasing incidence year by year and younger trend. Because the clinical symptoms of EC facilitate the disease to be detected timely, the majority of patients can be diagnosed in the early stages, and the prognosis is good, meanwhile, malignant degree of endometrioid adenocarcinoma is low,5 years survival rate is 90% or so. However, due to be sampled limitedly or misdiagnosed as irregular menstruation, about 15% patients had been in advanced stage of the disease stage, whose poor prognosis and mortality rate as high as 60%. the survival rate for a long period of time hasn’t been obviously improved, because of lack of new therapeutic methods, in addition to conventional operation, radiotherapy and chemotherapy. In recent years, tumor targeting therapy of cancer has become hotly and difficultly, there is no effective therapy direction targeting EC. Therefore, exploring new tumor target molecules of strong specificity, high sensitivity still has important clinical significance.NAD-dependent Class III histone deacetylase SIRT1 can deacetylate both histones and non-histone proteins. Its deacetylation enables it to interact with a variety of important transcription factors and transcriptional co-regulatory factors, to regulate gene transcription, chromosome stability and activity of target proteins. Thus it can participate in many kinds of biological functions such as energy metabolism, stress response, cell aging, therefore SIRT1 plays an important role in a variety of diseases, such as diabetes, cardiovascular disease, neurodegenerative diseases, metabolic and other diseases. The current results have displayed that SIRT1 plays a dual role as a tumor promoter as well as a tumor suppressor. Its involvement in tumorigenesis may be due to its diverse distribution in different tissues and different upstream and downstream regulatory factors that regulate its functions. SIRT1 controls lipolysis in adipocytes via FOXO1, and FoxO1 inhibits sterol regulatory element-binding protein-1c (SREBP-1c) gene expression via transcription factors Sp1 and SREBP-1c. There is no research at home and abroad about the correlation of SIRT1 and SREBP1.SREBP1 belongs to the family of the basic helix-loop-helix leucine zipper family of DNA binding transcription factors, which can regulate most enzymes involved in fatty acid biosynthesis, such as acetyl-CoA carboxylase, fatty acid synthase, Elovl-6, and stearoyl-CoA desaturase. Lipogenesis is increased in cancer cells, and the expression of SREBP1 has been observed to be elevated in various cancers, In our previous studies,we demonstrated that SREBP1 is overexpressed in endometrial and ovarian cancer, and the expression of SREBP1 protein increased with higher FIGO surgical stage and histological grade of the disease. Furthermore, knockdown of SREBP1 could induce apoptosis, reduce cell proliferation in vitro and in vivo. Meanwhile, Yanina Eberhard demonstrated that inhibition of SREBP1 or its downstream target fatty acid synthase sensitized resistant cells to death ligands. Therefore, SREBP1 plays a role as a cancer promoter in human cancers.Our aim was to investigate SIRT1 expression in endometrioid adenocarcinoma and its effect on tumor cells. Meanwhile, we too explored the correlation between SIRT1 and SREBP1.Accordingly, we proposed to establish the role of SIRT1 in EC.Part One SIRT1 expression in EC and its correlation with pathological featuresObjective:To detect SIRT1 protein, gene expression levels in endometrioid adenocarcinoma, atypical hyperplasia of endometrium and normal endometrium, and to analyze the correlation with SIRT1 expression levels and clinicopathological features. To preliminarily investigate the role of SIRT1 in occurrence, progression of endometrioid adenocarcinoma.Methods:Immunohistochemistry technique was used to detect SIRT1 expression in endometrioid adenocarcinoma, atypical hyperplasia of endometrium and normal endometrium, and through the follow-up study, we analysed the correlation between SIRT1 expression and clinical pathological parameters and the survival rate of the patients by the SPSS statistical analysis software. Fluorescence quantitative RT-PCR detection and Western-blot technology were used to detect SIRT1 expression levels in 60 cases of fresh endometrioid adenocarcinoma and adjacent normal endometrial tissues.Results:1. SIRT1 was expressed in the nucleus in endometrioid adenocarcinoma, atypical hyperplasia of endometrium and normal endometrium. The positive rates were 81.5%, 54.2%,47.1%. the positive rate in endometrioid adenocarcinoma was significantly higher than that in atypical hyperplasia of endometrium and normal endometrium, respectively P<0.05; there was no difference between atypical hyperplasia of endometrium and normal endometrium.2. The positive rate of SIRT1 expression in lower differentiation group was obviously higher than that in higher differentiation group, P<0.05; The positive rate of SIRT1 expression in stage III and IV group was significantly higher than that of stage Ⅰ and Ⅱ group, P<0.05; The positive rate in lymph node metastasis group was notablely higher than that without lymph node metastasis group, P<0.05; there was no difference between different age group, P>0.05.3. Survival analysis was performed by using the Kaplan-Meier method, the results showed as so, overall survival time of patients with positive SIRT1 expression was 34.68±3.80 months, that with negative SIRT1 expression was 44,93±3.31 months, overall survival time of patients with positive SIRT1 expression was apparently shorter than that with negative SIRT1 expression, P<0.05.4. SIRT1 gene expression in fresh endometrioid adenocarcinoma was significantly higher than that in the normal tissues adjacent to endometrioid adenocarcinoma, P<0.05; SIRT1 protein expression in fresh endometrioid adenocarcinoma was obviously higher than that in the normal tissues adjacent to endometrioid adenocarcinoma, P<0.05.Conclusion:1. SIRT1 expression was significantly increased in endometrioid adenocarcinoma compared to atypical hyperplasia of endometrium and normal endometrium.2. SIRT1 expression closely correlated with clinical pathological stage, histological differentiation, lymph node metastasis.3. The higher expression of SIRT1 was closely related to poorer prognosis of endometrioid adenocarcinoma.4. SIRT1 might play a role as a tumor promoter in endometrioid adenocarcinoma, and might be used as a new tumor marker for diagnosis and prognosis evaluation of endometrioid adenocarcinoma. notablely higher than that without lymph node metastasis group, P<0.05; there was no difference between different age group, P>0.05.3. Survival analysis was performed by using the Kaplan-Meier method, the results showed as so, overall survival time of patients with positive SIRT1 expression was 34.68±3.80 months, that with negative SIRT1 expression was 44,93±3.31 months, overall survival time of patients with positive SIRT1 expression was apparently shorter than that with negative SIRT1 expression, P<0.05.4. SIRT1 gene expression in fresh endometrioid adenocarcinoma was significantly higher than that in the normal tissues adjacent to endometrioid adenocarcinoma, P<0.05; SIRT1 protein expression in fresh endometrioid adenocarcinoma was obviously higher than that in the normal tissues adjacent to endometrioid adenocarcinoma, P<0.05.Conclusion:1. SIRT1 expression was significantly increased in endometrioid adenocarcinoma compared to atypical hyperplasia of endometrium and normal endometrium.2. SIRT1 expression closely correlated with clinical pathological stage, histological differentiation, lymph node metastasis.3. The higher expression of SIRT1 was closely related to poorer prognosis of endometrioid adenocarcinoma.4. SIRT1 might play a role as a tumor promoter in endometrioid adenocarcinoma, and might be used as a new tumor marker for diagnosis and prognosis evaluation of endometrioid adenocarcinoma.Part Two The influence and mechanism of SIRT1 on the biological behavior of endometrial cancer cellsObjective:To detect SIRT1 expression in endometrial carcinoma cell lines Ishikwa, ECC, RL95-2, KLE. By silencing SIRT1 expression with RNA interference technology, to dicuss the influence of SIRT1 on the cell growth, proliferation, migration and invasion of endometrial cancer cells, and to explore the mechanism of SIRT1 by detecting SREBP1 expression before and after the silence of SIRT1.Methods:Western-blot technology was used to detect SIRT1 expression in endometrial carcinoma cell lines Ishikwa, ECC, RL95-2, KLE; RNA interference technology was applied to silence SIRT1 expression,4 sets of SIRT1 SiRNA series were built and transducted into RL95-2 cells using lentiviral vectors; Western-blot technology was used to detect the transduction efficiency and SREBP1 expression.Results:1. The gray values of SIRT1 expression in endometrial carcinoma cell lines Ishikwa, ECC, RL95-2, KLE were 4834.87±105.68,5022.51±89.46,6769.22±121.07, 5822.68±178.64, it was visible that SIRT1 expression was higher in RL95-2 cell line.2. The gray values of SIRT1 expression in interference group and empty plasmid vector group were 1103.53±80.41,6157.70±212.69, transduction efficiency was more than 81.4%.3. Results of the automatic cell counting showed that:compared with empty plasmid group and blank control group, cell proliferation capability decreased obviously in interference group, P<0.05; however, there was no difference between empty plasmid group and blank control group.4. Results of the clone formation experiment showed that:the number of cell clone formation with diameter>10mm in interference group was significantly less than that in empty plasmid group and blank control group, P<0.05; however, there was no difference between empty plasmid group and blank control group.5. Results of Cell migration and invasion results showed that:the number of cell migration in interference group was significantly less than that in empty plasmid group and blank control group, respectively P<0.05; the number of cell invasion in interference group was significantly less than the empty plasmid group and blank control group, respectively P<0.05; there was no difference between empty plasmid group and.6. The gray values of SREBP1 expression in interference group and blank control group were 4815.68±101.28、2312.17±89.15, SREBP1 expression in interference group was lower than that of empty plasmid group, P<0.05.Conclusion:1. We had furtherly confirmed close correlation between SIRT1 expression and histological differentiation in EC, and selected the desired cell line that would be interfered.2. SIRT1 might play a role as a tumor promoter in EC, it could promote tumor cell growth, proliferation, migration and invasion.3. SIRT1 could be used as target sites for gene therapy, which provided new idea for the treatment of EC.4. SIRT1 could affected SREBP1 expression in EC, thus we Speculated that SIRT1 might play its role that could promote tumors by influencing SREBP1 and lipid metabolism, which need to be studied furtherly.