The Tumor-promoting Effect And Mechanism Of Glycerophospholipid-related Gene LPCAT1 In Endometrial Cancer

BackgroundEndometrial cancer(EC)is the second most common pelvic gynecologic malignancy in the world and the most common in the United States and Europe,affecting and claiming the lives of many women.EC usually occurs in women with metabolic syndrome and obesity.The incidence of EC increases with the increase of obesity.Studies have shown that the risk of EC increases by more than 50 percent for every five-unit increase in body mass index(BMI).The metabolic reprogramming of endometrial tumor cells after malignant transformation is a key process to adapt to the tumor microenvironment and maintain the malignant proliferative phenotype,and is also a key factor of tumorigenesis.Metabolic disorders,including enhanced glycolysis under anaerobic conditions,activation of lipid de novo synthesis pathway,and transformation of cellular energy supply pathway,are closely related to the prognosis of EC patients.In recent years,new therapeutic targets and new therapies have shown encouraging results in prolonging survival in EC patients,but morbidity and mortality in endometrial cancer remain unsatisfactory.Therefore,the study of targeted metabolic changes in the treatment of endometrial cancer is of great significance.Glycerophospholipids(GPLs)is the basic component of the biomembrane system.Glycerophospholipid metabolism is one of the most important processes in maintaining homeostasis.Enhanced glycerophospholipid synthesis provides sufficient membrane structure and energy source for rapidly proliferating cells in nutrient deficient conditions.There are increasing evidences that genes involved in the synthesis and remodeling of glycerophospholipids are involved in the development of cancer.However,the role of glycerophospholipid-related genes in the development and progression of endometrial carcinoma is rarely studied.The tumor microenvironment is an ecosystem composed of mesenchymal cells,immune cells,extracellular matrix(ECM)molecules and inflammatory mediators.These components have important influence on tumor development and clinical outcome.The major components of the tumor microenvironment include not only cancer cells but also immune cells and stromal cells.Studies have shown a significant correlation between tumor purity calculated by immunoscore and stromal score and clinical characteristics and prognosis of EC.In addition,other studies have shown that immune-related genes can be used as indicators of EC prognosis.Lipid metabolites are important factors affecting the tumor microenvironment,but it is largely unclear whether glycerophospholipid-related genes affect the EC microenvironment and clinical outcomes.In this study,bioinformatic methods were used to screen out GPLs-related metabolic genes by using TCGA database and we found that LPCAT1 can affect EC prognosis and EC tumor microenvironment.LPCAT1,lysophosphatidylcholine acyltransferase 1,is a phospholipid synthase with acyltransferase activity.A large number of studies have shown that LPCAT1 is highly expressed in breast cancer,lung cancer,glioblastoma and other malignant tumors and plays a carcinogenic role by regulating the intracellular phospholipid components,promoting tumor proliferation,invasion and metastasis.However,the role and mechanism of LPCAT1 in endometrial cancer remain unclear.In order to explore the role of LPCAT1 in endometrial cancer,we confirmed the tumor promoting effect and mechanism of LPCAT1 through a large number of basic medical experiments and bioinformatic methods.These results suggest that LPCAT1 may be used as a marker for early diagnosis of endometrial cancer and a target for precision immunotherapy in the future.Purpose1.The TCGA database was used to analyze whether the glycerophospholipid-related genes could affect EC tumor microenvironment and serve as an indicator for EC prognosis.2.The screened glycerophospholipid-related prognostic gene LPCAT1 was analyzed to explore its main functions in endometrial cancer through basic experiments and multi-level functional enrichment analysis.Methods1.Through differential analysis of endometrial cancer data in TCGA database,the key glycerophospholipid-related genes that can judge EC prognosis were screened by univariate COX regression,LASSO regression,random Forest model and PPI network construction.2.Identification of key glycerophospholipid-related genes that influence the EC tumor microenvironment by ESTIMATE and ssGSEA analysis.3.Knockdown and overexpression LPC AT1 cell lines were constructed by small interference technology and virus transfection technology,and the effects of knockdown or overexpression of LPCAT1 on the proliferation of endometrial cancer cells were explored by MTT,colony forming,EdU staining and flow cell cycle apoptosis assay.4.The effects of LPCAT1 on stemness phenotype and invasion and migration ability of endometrial cancer cells were explored by Transwell invasion and migration assay and cell sphere-forming assay.Western blot assay was used to detect the expression of molecules related to invasion and migration phenotypes and stemness phenotypes,and to confirm the molecular mechanism of the effect of LPCAT1 on the stemness phenotype and invasion and migration ability of endometrial cancer cells.5.The expression of key molecules in the TGF-β-Smad signaling pathway was detected by Western blot to explore the internal regulatory mechanism of LPCAT1 affecting the stemness transformation and invasion and migration phenotype of endometrial cancer cells.6.An animal model of endometrial cancer xenograft tumor was constructed using stabilized lentiviral cells.Tumor growth curve was plotted,tumor size was measured,and protein expressions of LPCAT1 and TGF-β-Smad signaling pathway were detected to explore the effects of LPCAT1 on malignant phenotype and molecular mechanism of endometrial cancer cells in vivo.7.The changes of metabolic substance content in endometrial cancer cells after overexpression of LPCAT1 were detected by liquid chromatography-mass spectrometry(LCMS)to explore the key lipid metabolites regulated by LPCAT1.Results1.Through differential analysis of endometrial cancer data in TCGA database,we found that 23 of 77 GPLs-related genes were differentially expressed in endometrial cancer,and 11 of them were related to prognosis of endometrial cancer.Four key genes,PLA2G2F,PLA2G2A,LPCAT2 and LPCAT1 were identified by LASSO model,random forest model and PPI network construction.2.The expression data are used with the ESTIMATE algorithm to estimate the stromal and immune cell content in malignant tumor tissue,predict the immune score and stromal score,and predict the tumor purity of each tumor sample.We found that among the four key genes,LPCAT1 is most closely related to the tumor microenvironment of endometrial cancer.The high LPCAT1 expression group had lower immune and stromal scores,lower ESTIMATE scores which related to higher tumor purity.Moreover,the expression of LPCAT1 was correlated with the content of 15 kinds of immune cells by differential analysis and correlation analysis.3.Through TCGA data analysis,we found that LPCAT1 expression was increased in endometrial cancer tissues,and overall survival,progression-free survival and disease-related survival were significantly shorter in the group with high LPCAT1 expression than in the group with low LPCAT1 expression.The expression of LPCAT1 was different in different tissue types,namely endometrioid carcinoma and serous carcinoma.Meanwhile,the expression of LPCAT1 increased with the increase of pathological grade.According to the area under ROC curve,LPCAT1 has a good diagnostic effect on endometrial carcinoma.Further immunohistochemical analysis of endometrioid adenocarcinoma tissues collected from Qilu Hospital confirmed the high expression of LPCAT1 in endometrial cancer tissues.4.In vitro MTT assay,colony forming assay,EdU assay and flow cell cycle apoptosis assay demonstrated that inhibition of LPCAT1 could inhibit the proliferation of endometrial cancer cells,while overexpression of LPCAT1 could promote the proliferation of endometrial cancer cells.Transwell chamber experiment proved that inhibition of LPCAT1 expression could inhibit the invasion and migration ability of LPCAT1,while overexpression of LPCAT1 could enhance the invasion and migration ability of endometrial cancer cells.Western blot experiment confirmed that after overexpression of LPCAT1,the expression of invasion and migration related molecules ZO-1 and Vimentin was significantly increased.Sphere forming experiments proved that overexpression of LPCAT1 significantly enhanced the sphere-forming ability of endometrial cancer cells.Western blot experiments confirmed that overexpression of LPCAT1 significantly increased the protein expression levels of stemness related transcription factors Nanog,SOX2 and OCT-3/4.5.We used second-generation RNA sequencing to analyze changes in gene expression in endometrial cancer cells after overexpression of LPCAT1,and found that TGFβ signaling pathway was significantly enriched by GSEA analysis.Western blot assay confirmed that protein expression levels of TGFβ1 and P-Smad were significantly increased after overexpression of LPCAT1.6.By using broad spectrum lipid metabolomics and liquid chromatography mass spectrometry,45 lipid metabolites with significant differences were screened out,including 24 down-regulated lipid metabolites and 21 up-regulated lipid metabolites.Phosphatidylethanolamine and phosphatidylcholine were significantly up-regulated.Lysophosphatidylcholine and triglyceride were significantly down-regulated in lipid metabolism.KEGG analysis suggested that these differential metabolites may be involved in two disease pathways in humans:insulin resistance and choline metabolism in tumors.Conclusion1.Compared with normal endometrial tissues,LPCAT1 is highly expressed in endometrioid adenocarcinoma tissues,and has good diagnostic and prognostic value.2.The expression level of LPCAT1 affects the purity of tumor cells and the proportion of immune cells in the tumor microenvironment of endometrial cancer.3.LPCAT1 can significantly promote the proliferation,invasion and migration of endometrial cancer cells;Meanwhile,LPCAT1 can increase the dryness of endometrial cancer cells.4.In endometrial carcinoma,LPCAT1 plays a carcinogenic role by activating TGF-βSmad signaling pathway.5.LPCAT1 mainly changes the contents of phosphatidylethanolamine and phosphatidylcholine in cells to play a metabolic reprogramming role.Innovations and LimitationsInnovations1.The role of LPCAT1 in regulating cell proliferation,migration,invasion and dryness in endometrial carcinoma was studied.2.LPCAT1 was found to affect the tumor microenvironment of endometrial cancer.3.It is clear that LPCAT1 promotes the occurrence and development of endometrial cancer through the activation of TGF-β-Smad signaling pathway4.The main lipid metabolites regulated by LPCAT1 in endometrial cancer cells were found.Limitations1.After LPCAT1 was overexpressed or knocked down,the co-culture of endometrial cancer cells and immune cells and the detection of related immune indicators were not performed,and the specific mechanism of LPCAT1 affecting the tumor microenvironment of endometrial cancer was not demonstrated.2.No rat tail vein injection was performed in vivo,and no data of lung metastasis model was obtained.3.There is no rescue experiment for metabolite changes regulated by LPCAT1 and TGFβ-Smad signaling pathway,and the demonstration is not complete.