Mechanism Of MGLL Induced Progression And Progesterone Resistance By Activating ROS/AKR1C1 Pathway In Endometrial Adenocarcinoma

Endometrial cancer(EC)is a common gynecological malignant tumor,with the incidence ranking the fourth and the mortality ranking the sixth among female malignant tumors.With the increasing incidence of Endometrial adenocarcinoma(EAC)and the younger age of patients,conservative treatment with progesterone has become an important choice for patients with fertility preservation.However,PR resistance is a major challenge in conservative treatment.There is an urgent need to clarify the molecular mechanism of malignant transformation and PR resistance of endometrioid adenocarcinoma,so as to target reverse the occurrence and development of EAC and PR resistance,and provide a solid guarantee for conservative treatment.Monoacylglycerol lipase(MGLL),a protein encoded by the MAGL gene,is a member of the serine hydrolase superfamily.It is mainly involved in fat mobilization and is the key enzyme in the final step of Triglyceride(TG)decomposition.TG was decomposed into Free Fatty Acids(FFAs)and glycerol.MGLL is highly expressed in invasive tumor cells such as colon cancer,ovarian cancer and breast cancer,and promotes the proliferation,migration and invasion of tumor cells by regulating tumor fatty acid network through MGLL-FFAs pathway.Knockdown of MGLL expression with inhibitors and shRNA reduced the synthesis of FFAs and inhibited the proliferation,migration and invasion of tumor cells.However,after administration of exogenous FFAs,the invasion of tumor cells was significantly enhanced,indicating that MGLL affects the invasion of tumor by regulating the level of FFAs.There was no significant difference in the expression of Fatty Acid Synthase(FASN)between invasive and non-invasive tumors.The administration of FASN inhibitors did not reduce the level of FFAs in cells,while the administration of fatty acid oxidation inhibitors significantly improved the proliferation,migration and invasion of triple-negative breast cancer cells.MGLL can directly promote fatty acid oxidation,which is the main energy pathway to maintain the invasion ability of highly malignant tumor cells.However,the expression of MGLL in endometrioid adenocarcinoma and its relationship with progesterone resistance remain unclear.This study is divided into three parts:First,through the analysis of second-generation transcriptome sequencing of progesterone-sensitive cell lines and resistant cell lines of endometrioid adenocarcinoma,determine progesterone-resistance-associated molecule MGLL and verify at the cell line protein level and histological level,and further through the database for the analysis of expression of endometrioid adenocarcinoma and adjacent non-cancerous tissues,determine that MGLL is correlated with tumor progression and progesterone resistance;continue to carry out the effect of different expression levels of MGLL on tumor progression in endometrioid adenocarcinoma tumor cell lines,and reveal the specific mechanism of its action through second-generation sequencing;finally verify the effect of different expression levels of MGLL on progestogen sensitivity in progestogen-sensitive and drug-resistant cell lines,and further explore the effect of MGLL inhibitor ABX-1431 in reversing progesterone resistance.Part Ⅰ Expression and significance of MGLL in endometrioid adenocarcinoma and progesterone resistant samplesObjective:1.To detect the expression of MGLL in progestin-sensitive and progestin-resistant endometrioid adenocarcinoma.2.To detect the expression of MGLL in endometrioid adenocarcinoma and adjacent non-cancerous samples.3.To explore the relationship between MGLL and the occurrence and development of endometrioid adenocarcinoma and progesterone resistance.Methods:1.To detect the expression of MGLL in progesterone-sensitive cell line-Ishikawa and progesterone-resistant cell line-IshikawaMR of human endometrioid adenocarcinoma:By analysing the second generation sequencing results of progesterone-sensitive cell line and resistant cell line of human endometrioid adenocarcinoma,to explore the expression of MGLL mRNA levels in the two cell lines;by WB experiment to verify the expression of MGLL protein levels in the two cell lines.2.To detect the expression of MGLL in progesterone-sensitive and progesterone-resistant clinical tissues of human endometrioid adenocarcinoma:Clinical tissue samples were collected from patients with endometrioid adenocarcinoma before and after conservative progestogen treatment,and the expression of MGLL in them was detected by IHC assay.3.To detect the expression of MGLL in human endometrioid adenocarcinoma and adjacent non-cancerous tissues:Analyze the expression of MGLL in matched endometrioid adenocarcinoma tumors and adjacent non-cancerous tissues in GEO database;collect the tumor tissues and adjacent non-cancerous tissues of patients with endometrioid adenocarcinoma,and detect the expression of MGLL by qRT-PCR assay and IHC assay respectively.Results:1.MGLL is highly expressed in progesterone-resistant endometrioid adenocarcinoma cell lines:The expression of MGLL in progesterone-resistant cell lines is significantly increased compared with progesterone-sensitive cell lines at the mRNA level;the expression of MGLL in progesterone-resistant cell lines is increased by about 300-fold compared with sensitive cell lines at the protein level.2.MGLL is highly expressed in progesterone-resistant endometrioid adenocarcinoma tissues:The expression of MGLL is significantly increased in samples from patients who fail to respond to progesterone therapy compared with samples from patients who achieve complete remission or partial remission after treatment,and MGLL tends to increase after progesterone therapy in the resistance group.3.MGLL is highly expressed in endometrioid adenocarcinoma:Bioinformatics analysis showed that the expression of MGLL expression in paired samples of endometrioid adenocarcinoma in GEO database was significantly increased in tumor tissues compared with adjacent non-cancerous samples;Experiments revealed that the expression of MGLL in endometrioid adenocarcinoma tumor tissue samples was significantly increased compared with adjacent non-cancerous samples.Conclusion:1.MGLL is highly expressed in progesterone-resistant endometrioid adenocarcinoma tissues and cell lines.2.MGLL is highly expressed in endometrioid adenocarcinoma.3.MGLL is associated with the progression of endometrioid adenocarcinoma and the development of progesterone resistance.Part Ⅱ The role and mechanism of MGLL in the development of endometrioid adenocarcinomaObjective:1.To investigate the effect of different MGLL expression on the malignant biological behavior of human endometrioid adenocarcinoma cell lines in vitro.2.Explore and verify the mechanism of MGLL-induced development of endometrioid adenocarcinoma.Methods:1.To detect the effect of MGLL overexpression on the malignant biological behavior of endometrioid adenocarcinoma cell lines:AN3CA cells with low MGLL expression level in human endometrioid adenocarcinoma cell lines were selected to construct MGLL overexpression and control cell lines,and the effect of MGLL overexpression on the malignant biological behavior of human endometrioid adenocarcinoma cell lines was verified by MTT,EDU,apoptosis,invasion and migration and WB experiments.2.To detect the effect of MGLL knockdown on the malignant biological behavior of endometrioid adenocarcinoma cell lines:HEC-1A cells with high MGLL expression level in human endometrioid adenocarcinoma cell lines were selected to construct MGLL knockdown and control cell lines,and the effect of knockdown of MGLL on the malignant biological behavior of human endometrioid adenocarcinoma cell lines was verified by MTT,EDU,apoptosis,invasion and migration and WB experiments.3.To screen and validate the molecular pathways of MGLL-induced endometrioid adenocarcinoma development:By transcriptome sequencing of MGLL-knockdown and control cells,differentially expressed genes were screened and enriched,and it was preliminarily speculated that MGLL may induce the development of endometrioid adenocarcinoma through ROS/AKR1C1 pathway.The sequencing results were verified by WB experiment and immunofluorescence experiment,and the recovery experiment was verified.Results:1.Overexpression of MGLL could promote the proliferation,inhibit apoptosis and promote metastasis of endometrioid adenocarcinoma cells:WB and qRT-PCR experiments verified the overexpression efficiency of MGLL,and in vitro experiments such as MTT,EDU,apoptosis,Transwell,and WB demonstrated that overexpression of MGLL can enhance the proliferation and migration of human endometrioid adenocarcinoma cell lines and reduce the proportion of apoptotic cells.2.Knockdown of MGLL could inhibit the proliferation,promote apoptosis and inhibit metastasis of endometrioid adenocarcinoma cells:WB and qRT-PCR experiments verified the knockdown efficiency of MGLL,and in vitro experiments such as MTT,EDU,apoptosis,Transwell,and WB demonstrated that knockdown of MGLL can weaken the proliferation and migration ability of human endometrioid adenocarcinoma cell lines and increase the proportion of apoptotic cells.3.MGLL promotes the progression of endometrioid adenocarcinoma by activating ROS/AKR1C1 pathway:GO and KEGG enrichment were performed on the transcriptome sequencing results of knockdown MGLL cell lines,and it was found that MGLL may regulate the expression of AKR1C1 and change the intracellular hypoxic microenvironment;IF assay and WB assay revealed that MGLL could regulate the expression of AKR1C1 by promoting the generation of ROS;recovery assay was performed to verify that MGLL promoted the occurrence and development of endometrial carcinoma by regulating ROS/AKR1C1 pathway.Conclusion:1.Overexpression of MGLL in endometrioid adenocarcinoma cell lines can enhance the malignant biological behavior of cells.2.Knockdown of MGLL in endometrioid adenocarcinoma cell lines attenuates the malignant biological behavior of the cells.3.MGLL promotes the progression of endometrioid adenocarcinoma by activating the ROS/AKR1C1 pathway.Part Ⅲ Role of MGLL in the development of progesterone-resistant endometrioid adenocarcinoma and effect of progesterone combined with ABX-1431Objective:1.To explore the changes of progesterone sensitivity in endometrioid adenocarcinoma cells with different MGLL expression levels.2.To explore the feasibility of reversing progesterone resistance in endometrioid adenocarcinoma by combining MGLL inhibitor ABX-1431 with progesterone.3.To verify that MGLL induces the development of progesterone resistance in endometrial adenocarcinoma by regulating the ROS/AKR1C1 pathway.Methods:1.To detect the changes of progesterone sensitivity of cells overexpressing MGLL in progestogen-sensitive cell lines of endometrioid adenocarcinoma:MGLL overexpression and control cell lines were constructed using Ishikawa,a progesterone-sensitive cell line of human endometrioid adenocarcinoma.After verifying the efficiency of constructing cell lines by WB assay and qRT-PCR assay,two cell lines were treated with MPA.The changes of progesterone sensitivity of overexpressed MGLL to endometrioid adenocarcinoma cell lines were verified by MTT,EDU,apoptosis,Transwell,WB and in vitro experiments and in vivo tumor formation in nude mice.2.To detect the changes of progesterone sensitivity of cells after knockdown of MGLL in progestogen-resistant cell lines of endometrioid adenocarcinoma:MGLL knockdown and control cell lines were constructed using human endometrioid adenocarcinoma progesterone-resistant cell line IshikawaMR.After verifying the efficiency of constructed cell lines by WB assay and qRT-PCR assay,two cell lines were treated with MPA.The changes of progesterone sensitivity of knockdown of MGLL in endometrioid adenocarcinoma cell lines were verified by MTT,EDU,apoptosis,Transwell,WB and in vitro experiments and in vivo tumor formation in nude mice.3.To detect the effect of MGLL-specific inhibitor ABX-1431 combined with progestogen in the treatment of progestogen resistance in endometrioid adenocarcinoma:The inhibitory effect of ABX-1431 on MGLL was verified by WB assay to determine the concentration used in subsequent assays;the in vitro killing effect of ABX-1431 combined with MPA was verified by MTT assay,colony formation assay,EDU assay,and flow apoptosis assay;and the in vivo killing effect of ABX-1431 combined with MPA was verified by tumor formation assay and IHC assay in nude mice.4.To verify that MGLL induces the development of progesterone resistance in endometrioid adenocarcinoma by regulating the expression of ROS/AKR1C1:By MTT assay and apoptosis assay to verify the changes in progesterone sensitivity of cells interfering with the expression of AKR1C1 in overexpressing MGLL cell lines.Results:1.Overexpression of MGLL reduces the sensitivity of endometrioid adenocarcinoma to progesterone and induces the occurrence of resistance:In the presence of MPA,overexpression of MGLL can enhance the proliferation and migration of progestogen-sensitive cell lines of endometrioid adenocarcinoma,reduce the proportion of apoptotic cells,and promote the growth of tumors compared with control cells.2.Knockdown of MGLL increases the sensitivity of endometrioid adenocarcinoma to progesterone reversing the occurrence of resistance:In the presence of MPA,knockdown of MGLL can weaken the proliferation and migration ability of progesterone-resistant cell lines of endometrioid adenocarcinoma,increase the proportion of apoptotic cells,and promote the growth of tumors compared with control cells.3.The killing effect of MGLL inhibitor ABX-1431 sensitizing progesterone on tumors:The combination of MGLL inhibitor ABX-1431 and MPA can significantly reduce the proliferation,promote apoptosis and inhibit the progression of progesterone-resistant cell lines of endometrioid adenocarcinoma.4.Interference with ROS/AKR1C1 pathway can reverse the malignant biological behavior of progesterone resistance caused by MGLL overexpression:In the presence of MPA,interference with AKR1C1 in cell lines overexpressing MGLL weakens the cell proliferation ability,increases the proportion of apoptotic cells,and enhances the sensitivity of cells to MPA.Conclusion:1.Overexpression of MGLL reduces the sensitivity of endometrioid adenocarcinoma to progesterone and induces the development of drug resistance.2.Knockdown of MGLL increases the sensitivity of endometrioid adenocarcinoma to progesterone and reverses the development of progesterone resistance.3.The combination of MGLL inhibitors ABX-1431 and progesterone can enhance the sensitivity of resistant cell lines to progesterone and enhance the killing effect of progesterone on tumors.4.MGLL induces the development of progesterone resistance in endometrioid adenocarcinoma by activating the ROS/AKR1C1 pathway.