A Study Of The Mechanism Of FTO Nucleus Transformation And Location Mediated By Estradiol In Estrogen Dependent Endometrial Cancer Cells

Endometrial cancer is the sixth leading cause of cancer?related death in women worldwide but the molecular mechanisms underlying its onset and progression are far from understood.It has proposed that there are two different clinicopathological types of endometrial carcinoma.Type I endometrial carcinoma comprises low?grade carcinomas,which make up more than 80% of all endometrial carcinomas.These tumors are often preceded by endometrial premalignant disease and are always estrogen nuclear receptor(ER)and progesterone receptor(PR)positive;they are often associated with a good prognosis.Type II endometrial carcinoma is typically represented by serous,clear?cell and mucinous adenocarcinoma.These tumors are represented by high histological grade carcinomas,arise from atrophic endometrium and appear to be estrogen?independent.Despite all these efforts and progresses observed in the past two decades,however,there is no perfect therapy effect for endometrial cancer.Surgery is common type of treatment for patients with endometrial cancer.Therefore,the molecular mechanism still remain unclear.Increase evidence support that obesity is a risk factor for endometrial cancer.The fat mass and obesity?associated(FTO)gene has been addressed overexpressed in endometrial tumor tissue,moreover,estrogen facilitated FTO nuclear localization and promoted endometrial cancer cell proliferation via estrogen nuclear receptor alpha signaling.On the other side,the estrogen membrane receptor GPR30 also overexpressed in endometrial cancer.Therefore,if it also mediates estrogen?driven FTO nuclear localization and promotes cell growth and invasion remains abscure.Block ERa signaling and isolation of cytoplasm or nucleus were performed to investigate the role of GPR30 on estrogen?driven FTO accumulation in nucleus.Immunoprecipitation(IP)was used to detect the binding between FTO and GPR30 proteins.The pretreatment with various inhibitors is to identify the signal pathway involved in FTO nuclear localization.Dual luciferase assay was used to determine the key sequence of FTO on FTO accumulation in nucleus.MTT assay and transwell assay were applied to detect endometrial cancer cell proliferative and invasive activation. We found that GPR30 mediated estrogen?driven FTO accumulation via mTOR signal pathway,which contributed to enhanced cell growth and invasion.Additionally,these functions were mediated by SSX8,a downstream gene of FTO.Thus,GPR30 mediated estrogen?driven FTO nuclear localization via mTOR signaling may be important in endometrial tumorigenesis and could serve as potential prognostic indicators and novel targets for therapeutic intervention.Therefore,this project has been investigated from following aspects: 1.Estrogen promote FTO nuclear localization by ER and GPR30;2.Which signal pathway involves in estrogen?driven FTO nuclear localization.3.The effects of FTO nuclear localization on endometrial cancer proliferation and invasion.Part ? estrogen promote FTO nuclear localization through ERa and GPR30 in endometrial cancerObjective: Obesity?related cancer can be partly explained by the imbalance of sex steroids,abnormal expression of insulin?like growth factors and oxidative stress.Therefore,the obesity related gene may play a role in endometrial cancer development.In previous studies,the fat mass and obesity?associated(FTO)gene was reported to be involved in cancer development.In this part,we applied western blot and IP to investigate the roles of ERa and GPR30 in estrogen?promoted FTO nuclear localization.Methods: Knockdown ERa was carried out by transfecting siRNA into endometrial cancer cells.After estrogen stimulation,western blot was used to determine the effect of ERa on FTO localization;On the hand,transfection of GPR30 was performed after estrogen stimulation,the FTO location also was detected by western blot.IP was used to examine the binding between FTO and GPR30.Result: Knock down of ERa resulted in decline of FTO accumulation in endometrial cancer cell nucleus.After blocking ERa signal pathway plus overexpression of GPR30 enhance estrogen?driven FTO nuclear localization.Conclusion: ERa and GPR30 play an essential role in estrogen?promoted FTO nuclear localization.Both receptors contribute to FTO nuclear localization.Therefore,our findings suggest that the estrogen receptor may play a role in endometrial cancer development.Part ? estrogen promote FTO nuclear localization through mTOR signal pathway in endometrial cancerObjective: Although less than 0.1% of the total cellular protein,kinase and multiple signal pathway play a pivotal role in conducting signals to control cell biofunction.In current part,we aim to investigate which signal pathway involves in estrogen-driven FTO nulear localization.Methods: Block ERa signaling and isolation of cytoplasm or nucleus were performed to investigate the role of GPR30 on estrogen-driven FTO accumulation in nucleus.The pretreatment with various inhibitors is to identify the signal pathway involved in FTO nuclear localization.Result: LY294002,the specific inhibitor of PI3K/AKT signal pathway blocked estrogen-induced FTO expression in cytoplasm,whereas accumulated FTO protein by estrogen in nuclear has been observed.Similar expression profiles by U0126,an inhibitor of ERK1/2 signal pathway,could be detected in endometrial cancer.rapmysin blocked estrogen-induced FTO expression not only in cytoplasm but also in nucleus,which suggests that mTOR signal pathway plays an essential role in estrogen-driven FTO nuclear localization.Conclusion: mTOR signal pathway plays an essential role in estrogen-driven FTO nuclear localization.Meanwhile,transfection of GPR30 plasmid plus estrogen stimulation significantly enhanced mTOR signal pathway activity.Part ? FTO nuclear localization enhance endometrial cancer cell proliferation and invasion and associated with poor clinical prognosisObjective: TO investigate the role of FTO on endometrial cancer cell proliferation and invasion.Meanwhile,explore its molecular mechanism and detect the association between its expression fashion and clinical prognosis.Methods: Construct the reporter gene and assay the role of different length of FTO fragment on FTO localization.Gene expression array was carried out to determine the downstream gene of FTO.MTT and transwell assay was performed to determine the effects of FTO downstream gene on endometrial cancer cell proliferation and invasion.Result: The full length of FTO gene is required for FTO nuclear localization.The SSX8 gene was identified as a downstream gene of FTO.Moreover,this protein affected endometrial cancer cell proliferation and invasion.The expression of FTO is associated with poor clinical outcome.Conclusion: FTO enhanced?proliferation and invasion is mediated by its downstream gene SSX8.This may provide a new therapy target gene in endometrial cancer administration.