The Role Of GIRM-19 In Regulating Autophagy And Glycolysis In The Pathogenesis Of Adenomyosis

ObjectiveAdenomyosis(AM)is a common gynecological syndrome in clinical gynecology.It usually occurs in women of reproductive period,most of them with the age ranged 30 from 50,there are also seen in young nulliparas.The clinical symptoms mostly are:menstrual disorders(40%-50%),prolonged menstruation and increased menstrual flow;dysmenorrhea(25%),characterized by secondary progressive dysmenorrhea.The treatment of adenomyosis should be fully based on the severity of patient’s symptoms,fertility requirements,etc.It hasn’t had radical treatment yet,mainly in use of expectant treatment,drug treatment and surgical treatment.As for the cause of adenomyosis,it is still unknown.Our study mainly discusses the role and molecular mechanism of the tumor suppressor gene GRIM-19 in the development of adenomyosis.We firstly examined the abnormal expression of GRIM-19 and the abnormal level of autophagy and glycolysis in tissue samples from patients with adenomyosis,and found the AMPK-ULK1 pathway associated with it,and then detected the activation of AMPK-ULK1 in adenomyosis patients.The regulation of GRIM-19 in the pathogenesis of adenomyosis by cell experiments and GRIM-19+/-mouse animal experiments have provided a powerful evidence in mechanism for the possible pathogenesis of adenomyosis.This study was based on the similar biological behavior of malignant tumors in adenomyosis and the abnormal metabolic level of eutopic endometrium,and the apoptosis-related gene GRIM-19 was involed in exploring the mechanism of GRIM-19 regulated autophagy and glycolysis in the eutopic endometrial tissues of adenomyosis,especially in metabolism of endometrial tissues,to elucidate the possible pathogenesis.MethodsOur study first examined the protein expression level of GRIM-19,AMPK,ULK1 and mRNA in endometrial and healthy control of patients with adenomyosis,and detected autophagy levels and glycolysis levels,and in the primary In the endometrial cells and the ISHIKAWA cell line,the mechanism by which GRIM-19 regulates autophagy and glycolysis via the AMPK-ULK1 signal transduction pathway was identified,and finally the GRIM-19+/-allele knockout mice were constructed in their uterus.Further verification in the organization.It mainly includes the following:1)Sterile collection of endometrial specimens meeting the experimental requirements,respectively,immersing each clinical specimen with physiological saline,and then preparing the sample to be tested(stored respectively in 4%paraformaldehyde solution or freeze at-80 ℃).2)The expression of GRIM-19,AMPK and ULK1 in each sample were detected by RT-PCR,immunohistochemical staining and Western blot technology,also detected the expression of autophagy markers Beclinl and LC3B and the key glycolytic enzyme PKM2 in order to evaluate autophagy and glycolysis level.3)The endometrial specimens of patients without adenomyosis group were collected,then isolated and cultured human primary endometrial cells of the tissues.The expression of GRIM-19 was up-regulated and down-regulated in the human primary endometrial cells by transfection technique.After that,the expression of AMPK and ULK1 in each sample was detected by Western blot,and the expression of autophagy markers Beclinl and LC3B were detected.4)The ISHIKAWA cell line was cultured in vitro,and the expression of GRIM-19 was up-regulated and down-regulated in the ISHIKAWA cell line by transfection technique,and then the expression of AMPK,ULK1 and autophagy-related proteins were detected,and the changes in cell viability were observed.5)The transfected ISHIKAWA cells were fixed with electron microscopy fixative,observed the cellular ultrastructure under electron microscope,and the formation of autophagosomes was observed to evaluate the level of autophagy.6)Construction of GRIM-19+/-allele knockout mice,sterile isolation of uterine tissue from GRIM-19+1-’allele knockout mice,simultaneous detection of AMPK,ULK1 and autophagy-related protein Beclinl and expression of LC3B.Results1.In the eutopic endometrial tissue of the control group and experimental group,GRIM-19 was mainly expressed in the cytoplasm of endometrial glandular cells.The expression level of GRIM-19 protein in the endometrial tissue of the experimental group was significantly lower than that of the control group(P<0.01).And there was no significant change in the different endometrial periodic(P>0.05).2.The level of autophagy in the eutopic endometrial tissues of adenomyosis was higher than that in the control group.The protein and mRNA expression level of autophagic marker Beclin1 in the experimental group was higher than that of the control group(P<0.01).The protein and mRNA expression of LC3B in experimental group was also higher than in the control group(P<0.01).The protein expression and mRNA expression of the key enzyme of glycolysis PKM2 was higher in experimental group(P<0.01),and these changes did not change with the endometrial periodic changes(P>0.05).3.1n the eutopic endometrial tissues of adenomyosis,AMPK and ULK1 were mostly localized in endometrial gland cells,and their protein expression and mRNA expression levels were significantly higher than those in the control group(P<0.01).There was no significant change in the eutopic endometrium of the control group and adenomyosis due to periodic changes in the endometrium(P>0.05).4.GRIM-19(transfected GRIM-19 plasmid)was up-regulated in ISHIKAWA cell line and human primary endometrial cells,AMPK,ULK1 protein expression and mRNA expression levels were significantly decreased compared with the control group(P<0.01),the level of autophagy was significantly lower than that of the blank control group(P<0.01).5.GRIM-19(transfected GRIM-19-siRNA)was down-regulated in ISHIKAWA cell line and human primary endometrial cells,then the expression of AMPK and ULK1 was significantly higher than that of the control group,and the expression level of autophagic maker Beclin1 and LC3B was also higher than that in the control group.(P<0.01).6.After up-regulating and down-regulating GRIM-19 expression in the ISHIKAWA cell line,autophagosomes were examined under electron microscopy to assess autophagy levels.7.The expression of AMPK and ULK1 protein in uterus of GRIM-19+/-allele knockout mice was higher than that of wild type control group(P<0.01),and the level of autophagy was also higher than that of wild type control group(P<0.01).Conclusions1.GRIM-19 is down-regulated in the eutopic endometrial tissue of patients with AM.2.Low-level expression of GRIM-19 may be involved in the regulation of autophagy and glycolysis in AM,thereby affecting the normal metabolism of AM in eutopic endometrium.3.GRIM-19 may regulate autophagy through the AMPK-ULK1 signal transduction pathway in endometrial tissues.