ENKUR Genes Inhibit The Proliferation And Invasion Of Endometrial Cancer Cells By Means Of Glucose Metabolism

BackgroundEndometrial cancer is a group of epithelial malignancies that occur in the endometrium,one of the three malignant tumors of the female reproductive tract.Endometrial carcinoma in recent years,the morbidity and mortality worldwide is on the rise,even in some developed countries,endometrial cancer becomes the first gynecological malignant tumor,which cause serious damage to women’s health.Studies have shown that the occurrence of endometrial cancer is closely related to the level of estrogen and progesterone,and is also related to abnormal activation of proto-oncogene and abnormal proliferation of cells.Some oncogenes promote the proliferation,invasion and metastasis of tumor cells by participating in the regulation of tumor cell metabolism.In recent years,with the development of molecular biology of tumor development,people have gradually realized the important role of energy metabolism reprogramming in tumor development.The occurrence and development of tumors is a multistep process under the action of physical factors,chemical factors and biological factors.In 1924,the famous biochemist Otto Heinrich Dr Warburg first found even in the case of sufficient oxygen,tumor cells in the glycolysis rate of more than two hundred times that of normal cells and tumor cells mainly glycolysis of tumor cell proliferation by a large amount of ATP.Glycolytic pathway of pyruvate is no longer to enter the Krebs cycle for aerobic oxidation,but under the catalysis of lactate dehydrogenase produce lactic acid,this phenomenon is defined as the Warburg effect of tumor cells.Although the Warburg effect mechanism have not been fully elucidated,but people have confirmed that some of the key regulatory mechanism and its closely related,such as mitochondrial oxidative phosphorylation damage,abnormal glucose metabolism enzyme expression,oncogene activation and tumor-suppressor gene inactivation and tumor microenvironment change,etc.Moreover,studies have shown that this metabolic reprogramming process is closely related to tumor cell proliferation and metastasis.The candidate gene ENKUR was selected in the early stage of this experiment to screen the interaction proteins through the yeast two-hybrid experiment.In this study,the endometrial cancer cell strain of ENKUR was established,and the role of ENKUR gene in endometrial cancer cells was understood through the function experiment of in vitro and in animals.We screened the interaction proteins of exogenous ENKUR through the co-ip experiment,and found that proteins involved in the metabolic pathways of multiple sugars were involved.The mechanism of ENKUR gene expression was explored by using immunofluorescence co-localization and Western Blot.Contents and methods1.The effect of ENKUR gene on endometrial carcinoma was studied in vitro.1)The endometrial cancer cell strain of ENKUR over expression was established,and the infection efficiency was detected by Western Blot.2)The proliferation ability of endometrial cancer cells after ENKUR over expression was detected by MTT,EDU and tablet cloning.3)Transwell and Boyden cell migration invasion test showed changes in invasion and metastasis of endometrial cancer cells after ENKUR over expression.2.Study on the effect of ENKUR gene on endometrial cancer in vivo animal level.1)The changes of endometrial cancer cells after ENKUR over expression were tested by the subcutaneous tumor experiment in nude mice.2)The effect of ENKUR gene on the invasion and metastasis of endometrial carcinoma in animals was detected by the rat hepatic envelope transfer model.3.A preliminary study on the molecular mechanism of ENKUR gene1)COIP test screened the interaction protein of ENKUR gene corresponding protein ENKURIN,and the immunofluorescence co-localization experiment was further verified in cells.2)Western Blot detected changes in glucose metabolism signaling pathway in endometrial cancer cells after ENKUR gene expression.3)Western Blot detected changes in the proliferation and EMT signaling pathways in endometrial cancer cells after ENKUR gene expression.4.The relationship between PGK1 and GRP78 and the clinicopathologic features and prognosis of endometrial cancer patients1)To collect 30 samples of normal endometrial tissue and 130 cases of endometrial carcinoma and pathological tissue specimens and clinical information of patients2)Immunohistochemical method was used to detect the expression of PGK1 and GRP78 in normal endometrial tissue and endometrial carcinoma3)Statistical analysis of the expression of PGK1 and GRP 78 was related to the clinicopathologic features and prognosis of endometrial cancer patientsResults1.The effect of ENKUR gene on endometrial carcinoma was studied in vitro.1)MTT,tablet clone,and EDU experiments confirmed that the proliferation ability of endometrial cancer cells was inhibited after ENKUR over expression.2)Transwell and Boyden experiments confirmed that the migration and invasion ability of endometrial cancer cells were inhibited after ENKUR over expression.2.Study on the effect of ENKUR gene on endometrial cancer in vivo animal level.1)The experiment with subcutaneous tumor of nude mice confirmed that the proliferation ability of endometrial cancer cells was inhibited after ENKUR over expression.2)The rat hepatic envelope metastasis model confirmed that the invasion and metastasis ability of endometrial cancer cells was inhibited after ENKUR over expression.3.A preliminary study on the molecular mechanism of ENKUR gene1)Co-immuno precipitatition(COIP)was used to screen the interaction protein of exogenous ENKURIN protein in endometrial cancer cells,and the results of mass spectrometry were 754 proteins.Some high score of proteins was selected,combined with COIP and Western Blot for validation,and ENKUR was found to interact with PGKl and GRP78.Cell immunofluorescence assay further confirmed that ENKUR and PGK1,PGK1 and GRP78 had co-localization in endometrial cancer cells.2)Western Blot experiment showed that after the stable overexpression of ENKUR,glucose metabolism related protein LDHA,GRP78,and beta-catenin were down-regulated,and the difference between GLUT1 and PGK1 was not obvious.3)Western Blot experiment showed that after the stable overexpression of ENKUR,EMT related protein E-cadherin up-regulated,N-cadherin,vimentin down-regulated,PI3K,p-PI3K,Akt,p-Akt down-regulated,cancer gene C-myc down-regulated,apoptosis related factors ATM,Bim,caspase9,caspase3 up-regulated.4.The relationship between PGK1 and GRP78 and the clinicopathologic features and prognosis of endometrial cancer patients1)PGK1 and GRP78 were highly expressed in endometrial carcinoma compared with normal endometrial tissues.2)The expression of PGK1 in endometrial carcinoma was different in different pathological types(P=0.007),histological grading(P=0.002),FIGO staging(P<0.001),and lymph node metastasis status(P<0.001).The expression of GRP78 in endometrial carcinoma was different from histological grade(P=0.019),FIGO staging(P<0.001),and lymph node metastasis(P<0.001).3)Survival curve analysis showed that the survival time of patients with PGK1 and GRP78 high expression was less than that of patients with low expression(P<0.001).ConclusionAfter the endometrial cancer cell line was stable over expression of ENKUR,the ability of cell proliferation,invasion and metastasis was weakened by a series of experiments in vitro and in vivo experiments.Screening of protein interaction of exogenous ENKUR by COIP experiment,combined with the result of the immunofluorescence positioning and Western Blot,we speculated that ENKUR may through interaction with PGK1,GRP78,and directly by GRP78,inhibition of sugar metabolism,thereby promoting apoptosis,inhibit the proliferation,invasion and metastasis of endometrial cancer cells.