Roles Of SIRT1/FoxO1/SREBP-1 In The Development Of Progestin Resistance In Endometrial Cancer

Background and objectiveEndometrial cancer(EC)is a group of epithelial malignant tumors that occur in the endometrium.It is one of the three major malignant tumors of the female reproductive tract,accounting for 7%of all malignant tumors in the female body and 20%to 30%of female reproductive tract malignancies.EC is the most common gynecologic malignancy in women in developed countries and is the fourth most common female malignancy.Its incidence is increasing year by year.According to statistics from the National Cancer Center,there were 63,400 new cases and 21800 deaths in China in 2015.A number of studies have found that progestin treatment is not only the main treatment for patients with early stage endometrioid adenocarcinoma that retains fertility,but is still effective for the treatment of patients with advanced and recurrent.The expression of progesterone receptor(PR)is found in endometrioid adenocarcinoma.Progestin inhibits the proliferation and invasion of EC cells through PR and promotes cell differentiation.PR can also act as a nuclear receptor involved in transcriptional regulation.PR-positive ECs have a higher response rate to medroxyprogesterone acetate(MPA)than PR-negative patients,suggesting that progestin exerts therapeutic effect through PR.However,there are still some patients with progestin treatment resistance,leading to treatment failure,disease progression and metastasis.Currently,there are few reports on the molecular basis of progestin resistance,and the mechanism is still unclear.Our previous studies confirmed that Sirtuin 1(SIRT1)is upregulated in EC tissue and knockdown of SIRT1 in EC cell lines downregulates sterol regulatory element binding protein-1(SREBP-1);increased expression of Forkhead transcription factor 1(FoxO1)can inhibit the proliferation of EC cells and downregulate the expression of SREBP-1;SREBP-1 is significantly upregulated in EC tissue.SIRT1 plays a role in PR-mediated rapid response modulation.SIRT1 abnormalities may lead to decreased expression of PR and induce progestin resistance in endometrioid adenocarcinoma.This article aims to discuss the role of SIRTl/FoxOl/SREBP-1 pathway in the development of progestin resistance in EC and its relationship with PR.MethodsStable progestin-resistant Ishikawa cell line was established by MPA selection and induction,and MTT assay was performed to compare the inhibitory rate and IC50 of the original Ishikawa cell line and progestin-resistant Ishikawa cell line.Flow cytometry compares the effect of MPA on the cell cycle distribution and apoptosis of the original Ishikawa cell line and progestin-resistant Ishikawa cell line;Transwell invasion assay compares the invasive ability of the two cell lines.Western-blot and real-time quantitative PCR were used to detect the expression of SIRT1,PR,FoxO1 and SREBP-1 in the original Ishikawa cells and progestin-resistant Ishikawa cells,and the change of molecular expression after MPA treatment.In the progestin-resistant Ishikawa cell line,SIRT1 was knocked down by lentivirus transfection,cell proliferation was detected by MTT assay,and the expression of the molecules was detected by Western-blot and real-time quantitative PCR.The statistical analysis was performed using GraphPad Prism version 5.Results were presented as mean±SD and analyzed using Student's t-test and two-way ANOVA assay.A probability(p)value<0.05 was considered to be statistically significant.All the experiments were performed in triplicate.Results(1)Progestin-resistant Ishikawa cells exhibited significant anti-MPA resistance in cell proliferation,cell cycle distribution,and apoptosis,and cell invasion was significantly enhanced(P<0.05).(2)At the protein and mRNA levels,the expression of SIRT1 and SREBP-1 increased in progestin-resistant Ishikawa cell line,and the expression of PR and FoxO1 decreased.After treated with MPA,the expression of SIRT1,SREBP-1,and PR decreased and the expression of FoxO1 increased in the original Ishikawa cell line,while MPA had no significant effect on progestin-resistant Ishikawa cell line.The above results were statistically different(P<0.05).(3)After knockdown of SIRT1 by lentivirus transfection,progestin-resistant Ishikawa cell line was more sensitive to the inhibitory effect of MPA and drug resistance was reduced.At the protein and mRNA levels,the expression of PR and FoxO1 increased,and the expression of SREBP-1 decreased(P<0.05).Conclusions(1)The progestin-resistant Ishikawa cell line which was established by MPA selection and induction was not sensitive to the inhibitory effect of MPA,and its invasive capability increased.(2)SIRT1/FoxO1/SREBP-1 acted as a pathway and played an important role in the development of progestin resistance in EC by regulating the expression of PR.