A Preliminary Research On The Function Of MAGL And Its Mechanism In The Carcinogenesis Of Endometrial Cancer

BackgroundEndometrial cancer is one of the most common malignant tumors in gynecology,its incidence is gradually increasing worldwide,and the age of onset is getting younger and younger.Currently,endometrial cancer is divided into two types:estrogen-dependent(type Ⅰ)and non-estrogen-dependent(type Ⅱ).Type Ⅰ accounts for the majority of endometrial cancers,most of which are endometrial adenocarcinoma.Type Ⅱ endometrial carcinoma is rare,including serous carcinoma,clear cell carcinoma,carcinosarcoma,and so on.In addition to the traditional classification of endometrial cancer,endometrial cancer is currently divided into four molecular subtypes according to the cancer genome atlas(TCGA):POLE mutant,microsatellite unstable(MSI),CN-low and CN-high.Early endometrioid adenocarcinoma of FIGO has a relatively good prognosis through surgery and necessary adjuvant therapy.However,for patients with advanced or relapsed,the 5-year survival rate has not improved significantly in recent years.With the development of individualized treatment and targeted therapy research,the targeted drugs such as bevacizumab,everolimus and sirolimus have also been added to the US National Comprehensive Cancer New web guide of the current medication treatment of endometrial cancer,in addition to hormone therapy and conventional platinum,paclitaxel,cyclophosphamide chemotherapy drugs.Therefore,the research of the pathogenesis and regulatory process of endometrial cancer is helpful to identify new therapeutic targets and develop new therapeutic approaches,so as to improve the prognosis of patients with advanced or recurrent endometrial cancer.With the changes of lifestyle,dietary patterns,obesity has become a public health problem threatening human health.Statistical studies predict that by 2030,overweight and obese people will account for about 58%of the global population.O besity is the most important risk factor for endometrial cancer.It is estimated that 40%of new cases of endometrial cancer are associated with obesity,among which type Ⅰ endometrial cancer is the most closely related to obesity.Obesity is an abnormal manifestation of fat accumulation in the body and is closely related to disorders of lipid metabolism.The occurrence and development of tumors is a complicated biological process,and energy metabolism disorders have become a hotspot of various cancer research.The malignant phenotype of tumor cells is often accompanied by reprogramming of metabolic pathways,including abnormal changes in lipid metabolism.In recent years,monoacylglycerol lipase(MAGL)as an important metabolic enzyme has been paid more and more attention in the occurrence and development of cancer.Studies have shown that MAGL can promote the development of tumors by controlling the release of free fatty acids(FFAs)to regulate lipid molecular signaling pathways.Many studies have shown that MAGL is highly expressed in human cancer cells and primary tumors,and inhibition of its expression attenuates the growth,survival,migration and invasion of invasive breast cancer,ovarian cancer and malignant melanoma cancer cells.MAGL is a ubiquitous enzyme with a molecular weight of 33 kDa and is a member of the serine hydrolase superfamily.It is a key enzyme in the hydrolysis of triglycerides to produce FFAs in lipid metabolism.Previous studies have shown that MAGL plays an important role in pathophysiological processes such as inflammation,pain,neuroprotection,neurodegenerative diseases,metabolic disorders and cancer.In recent years,more and more scholars have begun to pay attention to the role of MAGL in lipid metabolism and tumorigenesis.It has been reported that MAGL has functional expression in many invasive tumors such as colorectal cancer,gastrointestinal stromal tumor,prostate cancer,neuroblastoma,nasopharyngeal carcinoma,liver cancer,malignant melanoma,ovarian cancer,and breast cancer.Therefore,the effect of MAGL on endometrial cancer has attracted our attention.So far,there is no research report on the specific role of MAGL in endometrial cancer and its related mechanisms.The purpose of this study was to investigate the expression of MAGL in endometrioid adenocarcinoma,and to analyze the correlation between its expression and clinicopathological factors such as age,clinical pathological stage,depth of myometrial invasion,lymph node metastasis,BMI,lymphatic vascular space infiltrates and so on,and to calculate its correlation with the survival and prognosis of patients.Through the application of high-efficiency MAGL inhibitor JZL184 and siRNA in cell experiments to down-regulate the expression of MAGL,the effects of MAGL on the biological behavior of endometrial cancer cells were further explored from the protein level and gene level respectively,and the related mechanism was preliminarily explored,providing a new theoretical basis for whether MAGL could be a potential therapeutic target for endometrial cancer.Part One Expression of MAGL in endometrial cancer and its correlation with clinicopathological factorsObjective:To investigate the role of MAGL in the development of endometrial adenocarcinorna,the expression levels of MAGL in 28 cases of normal endometrium,31 cases atypical hyperplasia endometrium and 75 cases of endometrial carcinoma was detected.Then the expression of MAGL in endometrial cancer and its correlation with clinicopathological factors was analyzed.Methods:Immunohistochemistry was used to estimate the expression of MAGL in endometrial carcinoma,atypical hyperplasia endometrium and normal endometrial tissue in our research.GraphPad Prism 6.01 software was used to analyze the relationship between expression of MAGL and clinical pathological factors、the survival rate of patients.The quantitative expression of MAGL in 16 cases of fresh endometrioid adenocarcinoma tissues and corresponding adjacent normal endometrium tissues were detected by quantitative real-time RT-PCR.Results:1.The positive expression of MAGL in normal endometrium,atypical hyperplasia endometrium and endometrioid adenocarcinoma was manifested by brown staining of cytoplasm and cell membrane.The positive expression rate was 25.0%,51.6%and 76.0%correspondingly,and the differences in pairwise comparison were obviously.2.MAGL was highly expressed in endometrial carcinoma tissues,and its expression level was significantly correlated with FIGO stage,depth of myometrial invasion,number of pregnancies and BMI,P<0.05.However,histological grade,lymphatic metastasis,lymphatic vascular space infiltrates,age and with or without the combination of hypertension or diabetes had no correlation with expression of MAGL,P>0.05.3.The survival curve was drawn by Kaplan-Meier means,besides he survival rate was compared by Log-Rank time series test.The results showed that the survival rate of MAGL-positive group was lower than that of MAGL-negative,But the difference was not statistically significant,P>0.05.Patients with positive expression of MAGL had a poorer prognosis.4.The expression of MAGL mRNA in 16 pairs of fresh endometrial carcinoma tissues and corresponding adjacent normal tissues was detected by quantitative real-time RT-PCR,and the quantitative analysis of MAGL in endometrial carcinoma tissues was significantly higher than that in adjacent normal tissues,P<0.05.Conclusion:1.IHC and qRT-PCR researches showed that MAGL was highly expressed in endometrioid adenocarcinoma tissues.2.The expression level of MAGL in EC was significantly correlated with BMI,FIGO stage,depth of myometrial invasion and number of pregnancies3.MAGL might act as an oncogene in endometrioid adenocarcinoma,helping us to identify new therapeutic targets.Part Two The influence and preliminary mechanism of inhibiting the expression of MAGL on the biological behavior in endometrial cancer cellsObjective:The pharmacological inhibition and gene interference technology were used to down-regulate the expression of MAGL in endometrial cancer cells,and the influence and preliminary mechanism of MAGL on the biological behavior of endometrial cells were further investigated from protein and gene levels respectively.Methods:The expression of MAGL in endometrial cancer cells was inhibited from the protein and transcriptional leves by using JZL184 and siRNA respectively.The transduction efficiency was estimated by western-blot and qRT-PCR assays,and the effect on cell proliferation ability was detected by MTT.Transwell assay was used to detect the effect of MAGL inhibition on metastasis of tumor cells.The effects of MAGL down-regulation on tumor cell cycle and cell apoptosis were analysed by flow cytometry.To further explore the mechanism of MAGL in endometrial cancer cells,we evaluated the changes of Cyclin D1、Bcl-2、Bax and EMT correlation factors in protein levels and mRNA levels of endometrial cancer cells respectively again following MAGL inhibition.Results:1.We investigated the effect of drug JZL184 on endometrial cancer cells by exploring different gradient concentrations by MTT assay,and found that 1μmol/L JZL184 had the most significant effect on the proliferation of endometrial cancer cell lines.The JZL184 and siRNA were used to inhibit the expression of MAGL in endometrial cancer cells from protein and gene levels respectively,and the changes in final protein levels were detected by western blot.The effect of siRNA transfection on MAGL gene leve was estimated by qRT-PCR.It was found that the mRNA level of MAGL was significantly reduced,including 89.3%in Ishikawa,86.1%in RL95-2,and 74.7%in AN3CA respectively,with statistically significant differences.Protein levels of MAGL decreased by 70.6%in Ishikawa,70.3%in RL95-2,and 74.0%in AN3CA by JZL184 directly inhibite.Indirect inhibition of siRNA(mean siMAGLs)reduced protein levels of MAGL was 56.7%in Ishikawa,60.6%in RL95-2,and 66.1%in AN3CA.But no statistically significant difference was found between these two methods.2.MTT assay showed that both JZL184 and siRNA-mediated down-regulation of MAGL could inhibite the growth of endometrial cancer cells.The inhibition of cells treated with JZL184 is more unstable than that of siRNA,and its inhibition gradually decreases with time.However,there was no significant difference in the inhibition of cancer cell growth by the two methods.3.JZL184 and siRNA was used to knock down the expression of MAGL in endometrial cancer cells(Ishikawa,RL95-2),the transwell assay found that the migration and invasion ability of endometrial cancer cells was significantly inhibited,which suggested that MAGL was involved in the metastasis of endometrial cancer and played a certain role in the development of EC.4.Flow cytometry was performed to evaluate the changes of endometrial cancer cells after knocking down MAGL.It was found that both JZL184 inhibition and siRNA interference MAGL could induce endometrial cancer cell cycle arrest in G0/G1 phase compared with the control group,P<0.05.The G0/G1 phase of Ishikawa cells transfected with siMAGL increased by 20.6%,the RL95-2 increased by 41.5%,and AN3CA increased by 30.3%.In the cells treated with JZL184,the proportion of G0/G1 phase cells increased by 16.2%in Ishikawa cells,28.6%in RL95-2 cells and 11.8%in AN3CA cells.Apoptosis experiments of cells showed that the proportion of early and late apoptosis in Ishikawa cells transfected with siMAGL 1 was 6.9710.58%,and the proportion of early and late apoptotic cells in Ishikawa cells transfected with siMAGL2 was 10.27±1.19%,compared with the control group which was 5.38±0.08%,the difference was statistically significant.In Ishikawa cells treated with JZL184,the proportion of early and late apoptotic cells was 7.52±0.84%,which was significantly higher than 4.90±0.85%in the untreated group.The proportion of apoptosis of RL95-2 cells transfected with siMAGL1 was 9.17±2.29%,and siMAGL2 was 8.71±1.82%,which was both higher than the control group significantly,P<0.05.In RL95-2 cells treated with JZL184,the proportion of early and late apoptotic cells was 7.01±0.57%,which was significantly different from the 4.97±0.95%in the untreated group.The experimental results showed that inhibiting MAGL can induce apoptosis of EC cells.MAGL may play a vital role in the regulation of apoptosis of EC cells.5.After siRNA(mean siMAGLs)silenced MAGL of the endometrial cancer cell lines,both the levels of cyclin D1 and Bcl-2 genes were significantly decreased,which decreased Cyclin D1 in Ishikawa cells by 52.2%,RL95-2 cells by 65.9%and AN3CA cells by 49.8%.The transcription level of Bcl-2 was reduced by 59.6%in Ishikawa cells,67.4%in RL95-2 cells and 56.6%in AN3CA cells.The protein expression of cyclin D1 in Ishikawa cells was decreased by 33.2%in JZL184-treated endometrial cancer cells,34.4%in RL95-2 cells and 49.8%in AN3CA cells,while Bcl-2 protein levels decreased by 35.1%in Ishikawa cells,36.2%in RL95-2 cells and 29.3%in AN3CA cells.Bcl-2 and Bax are a pair of relatively important apoptosis regulatory proteins.The expression ratio of the two is an important factor of apoptotic.We used qRT-PCR and western blot to detect the mRNA and protein level of cells which have treated with siRNA and JZL184 respectively.We have confirmed that the level of Bcl-2 mRNA and protein expression in EC cells was significantly reduced after the inhibition of MAGL previously.Using the same method,we detected the expression of Bax.Compared to the control group,the mRNA levels of Bax have not changed significantly.But the mRNA level of bcl-2/bax in the intervention group of siRNA was reduced significantly.We also used western blot to measure the protein expression levels of bcl-2 and bax in EC cell lines.With the intervention of JZL184,the expression of bcl-2 protein was significantly reduced,consistent with the previous part of the experimental.While the bax protein was significantly higher than the control group,which was different from the mRNA level detected in siMAGL group.These findings implied that MAGL may regulate the apoptosis of EC cells by changing the expression of Bcl-2 and Bax.6.The expression of MAGL in endometrial cancer cells was interfered by siRNA(mean siMAGLs),then qRT-PCR showed that E-cadherin mRNA levels were significantly up-regulated,while Vimentin and snail gene expressions showed a significant downward trend.The protein expression of E-cadherin in Ishikawa and RL95-2 cells was significantly increased by JZL184.The protein expression of Vimentin and snail was consistent with the gene level,which showed a significant decrease.The inhibition of MAGL by SiRNA and JZL184 on MAGL can cause the change of EMT-related factors,and MAGL promoting the migration of endometrial cancer cells may be related to EMT-related factors.Conclusion:1.MAGL plays a role as an oncogene in endometrial cancer,and inhibiting its expression inhibits the proliferation and growth,metastasis and induces an increase in the number of cell apoptosis.2.MAGL may promote the growth,proliferation and inhibit apoptosis of tumor cells by regulating the CyclinD1、Bcl-2 and Bax.3.MAGL promotes metastasis of endometrial cancer cells may be related to EMT-related factors.4.MAGL provides new ideas for the treatment of endometrial cancer,which may become a new target of therapy for endometrial cancer.Part Three Effect of knocking down MAGL on the growth of transplanted tumors in nude miceObjective:Our experiments have proved that MAGL plays an important role in endometrial cancer with tissues and cells.In order to explore the effect of MAGL on the growth of endometrial cancer in vivo,the effect of knockdown of MAGL on the growth of endometrial cancer was determined by subcutaneous transplantation tumor experiments in nude mice.Methods:The transfection efficiency was verified by fluorescence and western blot experiments in Ishikawa cells transfecting with shMAGL lentivirus.Nude mice were divided into 4 groups randomly,shCtrl-Ishikawa,shMAGL-Ishikawa,Ishikawa and Ishikawa cells were inoculated for tumor formation experiments respectively to establish a nude mouse xenograft model of endometrial cancer.Observe the growth of subcutaneous tumors dynamically,measure the tumor volume and the weight of nude mice every other day after seeing the visible tumor.On the 15th day,the two groups inoculated with Ishikawa cells were injected intraperitoneal with JZL184(16mg/kg qod)and control solvent(physiological saline)equal volume respectively,both inject 10 times.The nude mice were sacrificed by cervical dislocation on the 33rd day,then the tumor entities were stripped,photographed and the tumor volumes was measured and recorded.Results:Western blot confirmed that MAGL expression in shMAGL group was significantly lower than in control group.The tumorigenicity was significantly different,and the tumor volume(448.2±69.1 mm3)significantly decreased compared with the control group(1199.3±228.0mm3)and was significantly inhibited after knocking down MAGL.Similarly,the growth of transplanted tumor in nude mice treated with JZL184(514.6±138.8 mm3)was slow,and there was a significant difference in growth trend compared with the control group(1283.5±203.9mm3)..The in vivo assays showed that the growth of subcutaneous transplanted tumor of nude mice could be inhibited by intervened with shMAGL and JZL184.Conclusion:In vivo experiments on tumor formation in nude mice have shown that the tumorigenicity of subcutaneously transplanted tumors in nude mice is significantly reduced after the inhibition of MAGL,the tumor growth is slow,and the tumor volume is significantly suppressed.Knock down of MAGL can inhibit the growth of subcutaneouse transplanted tumors in nude mice.