Mechanism Of SREBP1 Induced Progesterone Resistance By Activating NF-κB Pathway In Endometrial Adenocarcinoma

Endometrial carcinoma(EC)is the most common form of malignant gynaecological tumor,ranking in 4th and 6th place of all cancers with respect to morbidity and mortality,respectively.There is an apparent trend for younger patients to be affected by endometrial adenocarcinoma(EAC),these patients are normally inclined to select conservative forms of treatment so as to maintain their fertility.A phase II clinical trial revealed that approximately 55%of EAC patients showed a complete response to conservative forms of treatment that included medroxy-progesterone acetate(MPA),with a 47%recurrence rate between 7 and 36 months.Nevertheless,other studies have shown that in excess of 30%of EC patients do not respond to MPA and acquire resistance to progesterone during the course of progesterone treatment.Conservative progesterone-based treatment is also an effective treatment for advanced and recurrent endometrial cancer.To deeply explore the molecular mechanism of progesterone resistance in endometrial adenocarcinoma and improve progesterone resistance and poor prognosis has become an urgent clinical problem to be solved.Sterol regulatory element-binding proteins(SREBPs)are transcription factors that regulate the anabolism of cholesterol and lipids.SREBPs activate the transcription of target genes by binding with the sterol regulatory elements of the lipid synthase gene promoter or enhancer.In addition,it has also been reported that a reduction in the levels of sterols in tumor cells will cause SREBP1 to be synthesized into precursors within the endoplasmic reticulum,thus creating protein complexes with SREBP-cleavage-activating protein(SCAP).These complexes are then transported to the Golgi apparatus for hydrolysis,thus releasing SREBP1 into the nucleus and activating the transcription of enzymes related to cholesterol and fatty acid anabolism.When the levels of sterols increase,the insulin inducible gene(INSIG)will block the transportation of SREBPs-SCAP from the ER to the Golgi apparatus by binding with SCAP;this reduces the production of lipids and cholesterol.SREBP1 has been found to be abnormally expressed in a variety of human tumor,including prostate carcinoma,breast carcinoma,and hepatocellular carcinoma.In addition,SREBP1 is overexpressed in endometrial cancer,which is related to the degree of tumor tissue differentiation,and endogenous SREBP1 promotes the occurrence and development of endometrial cancer.This study is mainly divided into three parts of the experiment:First,we analyzed the possible involvement of SREBP1 in progesterone resistance in endometrial adenocarcinoma by verifying the expression of SREBP1 in progesterone-sensitive and-resistant tissue samples and cell lines of endometrial adenocarcinoma;Then,we confirmed the specific role of SREBP1 in the development of progesterone resistance in endometrial adenocarcinoma by exploring the changes in the sensitivity of different intracellular SREBP1 expression levels to progesterone,and used in vitro and in vivo experiments to explore the SREBP1 inhibitor Fatostatin to help reverse progesterone resistance;Finally,we clarified and validated the specific mechanism of progesterone resistance induced by SREBP1 in endometrial adenocarcinoma by bioinformatics and in vitro experiments.Part I The expression and significance of SREBP1 in progesterone resistance of Endometrial adenocarcinomaObjective:To detect the expression of SREBP1 in progesterone-sensitive and-resistant clinical samples and cell lines of endometrial adenocarcinoma and explore the correlation between SREBP1 and the occurrence of progesterone-resistant endometrial adenocarcinoma.Methods:1.To detect the sensitivity of human endometrial adenocarcinoma cells to progesterone:The sensitivity of progesterone-sensitive cell lines,primary progesterone-resistant cell lines and secondary progesterone-resistant cell lines to progesterone was detected by MTT assay,EDU incorporation assay and flow cytometry apoptosis assay,in order to determine the reliability of the cell lines used in this study.2.To detect the expression of SREBP1 in progesterone-sensitive and-resistant cell lines of endometrial adenocarcinoma:Download the gene expression microarray of progestin-sensitive cells and resistant cells by GEO,analyze the mRNA expression of SREBP1 in progesterone-sensitive and-resistant cells using R language;verify the expression level of SREBP1 in progesterone-sensitive,primary and secondary progesterone-resistant cell lines by Western blot assay,and clarify the changes of SREBP1 after using progesterone treatment.3.To detect the PGR expression in progesterone-resistant clinical samples:The PGR expression in collected clinical samples by IHC assay is associated with the clinical prognosis of patients to clarify the reliability of clinical samples used in this study.4.To detect SREBP1 expression in progesterone-resistant clinical samples:SREBP1 staining by IHC in clinical samples from patients with endometrial adenocarcinoma before and after conservative treatment with progesterone was detected.Results:1.Sensitivity of human endometrial adenocarcinoma cells to progesterone:Ishikawa,HEC-1A,and IshMR,cell lines were selected as progesterone sensitive cells,primary progesterone resistant cells,and secondary progesterone resistant cells,respectively,for subsequent experiments.The results showed that under the same concentration of MPA,compared with the progesterone-resistant cells IshMR and HEC-1A,the viability and proliferation of progesterone-sensitive cells Ishikawa were significantly decreased,apoptotic cells were significantly increased,and the sensitivity of Ishikawa to MPA was significantly higher than that of IshMR and HEC-1A.2.SREBP1 was highly expressed in progesterone-resistant cell lines of endometrial adenocarcinoma:The expression of SREBP1 in progesterone-resistant cell lines was increased compared with sensitive cell lines.After MPA treatment,the expression of SREBP1 in resistant cells was further increased.3.PGR barely expressed in progesterone-resistant clinical samples:IHC showed that PGR was significantly positive in CR and PR samples,and there was no significant difference before and after progesterone treatment,but barely express in the group with progressive disease.It was considered that the expression level of PGR was basically consistent with the results of clinical prognosis.4.SREBP1 positively expressed in progesterone-resistant clinical samples:IHC showed that SREBP1 expression was low in both CR and PR samples,while SREBP1 expression was positive in the disease progression group,and SREBP1 expression was further increased after treatment,with consistent nuclear staining results.Conclusion:1.SREBP1 is highly expressed in progesterone-resistant clinical specimens and progesterone-resistant cells of endometrial adenocarcinoma.2.SREBP1 is related to the occurrence and development of progesterone resistance in endometrial adenocarcinoma.Part Ⅱ Effect of SREBP1 on the development of progesterone resistance in endometrial adenocarcinomaObjective:1.To investigate the change of progesterone sensitivity of endometrial adenocarcinoma cells after targeted SREBP1 treatment.2.To investigate the feasibility of reversing progesterone resistance in endometrial adenocarcinoma using the SREBP1 inhibitor Fatostatin in combination with progesterone.Methods:1.To detect the malignant biological behavior and progesterone sensitivity of endometrial adenocarcinoma after SREBP1 overexpression:Progesterone-sensitive cells were used to construct SREBP1 overexpression and control cell lines.After treatment with different concentrations of progesterone,the effect of SREBP1 on the malignant biological behavior and the change to progesterone sensitivity of endometrial adenocarcinoma cell lines were verified by MTT,EDU,apoptosis,WB and tumor formation of mice experiments in vitro and in vivo.2.To detect the malignant biological behavior and progesterone sensitivity of endometrial cancer after SREBP1 suppression:Progesterone-resistant cells were used to construct SREBP1 suppression and control cell lines.After treatment with different concentrations of progesterone,the effect of SREBP1 on the malignant biological behavior and the change to progesterone sensitivity of endometrial adenocarcinoma cell lines were verified by MTT,EDU,apoptosis,WB and tumor formation of mice experiments in vitro and in vivo.3.To detect the effect of SREBP1 inhibitor combined with progesterone on malignant biological behavior of endometrial adenocarcinoma:After verifying the inhibitory effect of SREBP1 inhibitor by WB experiment,inhibitor combined with progesterone to act on progesterone-resistant cell lines to observe the development of tumors in vitro and in vivo experiments.Results:1.Overexpression SREBP1 could reduce the sensitivity of cells to progesterone and induce progesterone resistance in endometrial adenocarcinoma:After WB and PCR experiments verified the overexpression efficiency of SREBP1,in vitro experiments such as MTT,EDU,apoptosis,and WB and in vivo experiments in tumor formation demonstrated that SREBP1 overexpression could decrease the sensitivity of progesterone-sensitive cell lines to progesterone and tend to the biological behavior of progesterone-resistant cell lines.2.Suppression SREBP1 could increase the sensitivity of cells to progesterone and reverse progesterone resistance in endometrial adenocarcinoma:After WB and PCR experiments verified the suppression efficiency of SREBP1,in vitro experiments such as MTT,EDU,apoptosis,and WB and in vivo experiments in tumors demonstrated that SREBP1 interference could improve the sensitivity of primary and secondary progesterone-resistant cell lines to progesterone and tend to the biological behavior of progesterone-sensitive cell lines.3.The combination of SREBP1 inhibitor and progesterone increase the killing effect of progesterone on tumor:After verifying the inhibitory effect of SREBP1 inhibitor by WB assay,in vitro experiments such as MTT,clone formation and apoptosis experiments and in vivo experiments of tumor formation demonstrated that the combination of inhibitor and progesterone could significantly reduce cell proliferation,promote apoptosis and inhibit tumor progression.Conclusion:1.The overexpression of SREBP1 is capable of reducing the lethal effect of MPA on endometrial adenocarcinoma cells and induces the emergence of progesterone resistance.2.The suppression of SREBP1 in progesterone-resistant cells enhanced the sensitivity of cells to progesterone and reversed progesterone resistance.3.Fatostatin,a SREBP1 inhibitor,enhanced the sensitivity of endometrial adenocarcinoma to progesterone,thus inhibiting tumor growth.Part Ⅲ Mechanism of Progesterone Resistance Induced by SREBP1 in Endometrial adenocarcinomaObjective:To explore the mechanism underlying the effect of SREBP1 on progesterone resistance in endometrial adenocarcinoma and authenticate it reversely.Methods:1.To screen and verify that the molecular pathway of progesterone resistance induced by SREBP1 in endometrial adenocarcinoma:Whole transcriptome sequencing was used to screen differentially expressed genes between SREBP1 overexpression cell and control cell.GO and KEGG analysis were used to identify the signaling pathway.KEGG analysis result was verified by WB and immunofluorescence experiments.The possibility of SREBP1 activating the NF-κB pathway was further confirmed after progesterone treatment.2.To detect NF-κB expression in progesterone-resistant clinical samples:NF-κB staining by IHC in clinical samples from patients with endometrial adenocarcinoma before and after conservative treatment with progesterone was detected.3."Recovery experiment" with NF-κB knockdown:Small interfering RNA(siRNA)targeted NF-κB was used to knockdown the expression of NF-κB in overexpressing SREBP1 cell line and control group.After verifying the transfection efficiency by WB assay,the sensitivity of cells to progesterone was verified by MTT,apoptosis and other experiments,and the expression of proliferation and apoptosis-related proteins was detected by WB assay.Results:1.SREBP1 overexpression could activate NF-κB pathway:Differentially expressed genes between SREBP1 overexpression cell and control groups were and analyzed,and KEGG enrichment revealed that the NF-κB pathway was significantly enriched in overexpressing SREBP1 cells.Immunofluorescence and WB experiments revealed that overexpression of SREBP1 could activate NF-κB pathway and promote its nuclear translocation,and this phenomenon was more obvious after progesterone treatment.2.NF-κB was positively expressed in progesterone-resistant clinical samples:IHC experiments revealed that NF-κB barely express in both CR and PR groups,while NF-κB expression was increased in the PD/SD group,and the results were consistent with the trend of SREBP1 in cells.3.Inhibition of the NF-κB pathway could reverse the rEDUced progesterone sensitivity of cells induced by SREBP1 overexpression:The expression of NF-κB was knocked down by siRNA of NF-κB in SREBP1 overexpression cell lines and control group.MTT and apoptosis experiments were carried out.The results showed that NF-κB knockdown could improve the decrease of progesterone sensitivity induced by SREBP1 overexpression and reverse the progesterone resistance induced by SREBP1 overexpression.Conclusion:1.SREBP1 induced progesterone resistance by promoting the translocation of NF-κB to the nucleus and by activating the NF-κB pathway.2.Knockdown of NF-κB pathway can reverse the decreased sensitivity of cell lines to progesterone induced by SREBP1 overexpression reverse the occurrence of progesterone resistance.