{"paper_id":"e50347ce-5882-48a8-8409-a3f2a9ecef13","body_text":"www.ijpsonline.com\nSpecial Issue 2, 2020 Indian Journal of Pharmaceutical Sciences\n35\n*Address for correspondence\nE-mail: yanglina8498de@163.com\nDifferential Expression of ANXA2 in Eutopic and Ectopic \nEndometrium\nHAIYAN YU, QINGLING LU1, YITING WANG1, X. MA, BO LI1 AND LINA YANG1*\nHealth Management Center, 1Department of Gynecology, Heilongjiang Provincial Hospital, Harbin City, 150036, Heilongjiang \nProvince, China\nYu et al.: Differential Expression of ANXA2 in Eutopic and Ectopic Endometrium\nThe purpose of this study was to compare the differences in expression of annexin A2 between eutopic and \nectopic endometria and to explore the pathogenesis of adenomyosis. In this study, 22 adenomyosis patients \nwho underwent uterine laparotomy in Heilongjiang Provincial Hospital from April 2016 to October 2018 \nwere enrolled as the observation group. The eutopic and ectopic endometria were used for the experiment. \nIn addition, 22 uterine fibroids patients with normal endometrium who underwent open hysterectomy were \nselected as the control group. The levels of annexin A2 in endometrial tissue and serum were compared \nbetween the two groups. The results showed that the annexin A2 level in normal endometrial tissue was \nsignificantly lower than that in the eutopic and ectopic endometrial tissue and the annexin A2 level in the \nectopic endometrium was significantly higher than that in the eutopic endometrium (p<0.05). The annexin \nA2 level in the serum of the observation group was significantly (p<0.05) higher than that of the control \ngroup. Clinical monitoring of changes in annexin A2 level could be useful as auxiliary diagnostic marker to \nstart treatment of adenomyosis, so as to project prognostic changes more accurately. However, the results \nof this study still need to be verified with multi-center samples.\nKey words: Annexin A2, eutopic endometrium, ectopic endometrium, expression difference, adenomyosis\nAdenomyosis (AM) mainly refers to a type of disease in \nwhich benign infiltration and growth occur constantly \nin the glands or interstitium of the endometrium \ntowards the muscular layer, causing symptoms such as \ndysmenorrhea or excessive menstruation [1]. This is an \nestrogen-dependent disease that can be pathologically \nbenign. However, some biological behaviors, such as \nimplantation, infiltration and recurrence of malignant \ntumors have a great impact on patients’ quality of life[2-4]. \nDetection of serum CAl25 is helpful for diagnosis, but \nfinal diagnosis still requires surgery and patholog y. \nDue to the lack of reliable diagnostic indicators, it \nis inevitable to misdiagnose or miss the diagnosis. \nTherefore, it is of great significance to search for a \ndiagnostic marker. In addition, moderate and severe \ndysmenorrhea seriously affects the physical and mental \nhealth and quality of life of women of childbearing age \nbut the relevant mechanism is still not very clear and \nthe root cause urgently needs to be investigated.\nAnnexin A2, also known as Annexin II, AXII and \nANXA2. ANXA2 is more commonly expressed in \nhuman endothelial cells, neutrophils, bone marrow \n\nwww.ijpsonline.com\nSpecial Issue 2, 2020Indian Journal of Pharmaceutical Sciences\n36\ncells, brain cells and some tumor cells. It is highly \nexpressed in cells with high proliferation and \ndifferentiation ability, and is expressed in cells with \nlow differentiation. About 1/3rd of ANXA2 is produced \nby endothelial cells, which play a very important role \nin the cells in and out of the body [5]. Within the cell, \nit is responsible for cell proliferation, differentiation, \napoptosis, DNA synthesis, RNA binding, mRNA \ntransport, cytoskeletal remodeling and stabilization, \nbiofilm formation, vesicle trafficking. Outside the cell, \nit is responsible for osteoblast formation and bone \nresorption, channel formation of Ca 2+, opening and \nregulation of ion flux frequency, signal transduction, \nimmunoglobulin transport, cell metastasis and invasion, \nepithelial mesenchymal transition (EMT), fibrinolysis, \nand the formation of new blood vessels. And in many \ndiseases, such as human immunodeficiency virus \n(HIV), hepatitis B, hepatitis C, diabetic nephropathy, \nthyroid disease, sepsis, endometriosis, melanoma, \npsoriasis, keloid, acute young granulocyte leukemia, \nlymphoma, multiple myeloma, acute promyelocytic \nleukemia, lupus nephritis and malignant tumor, it also \nplays a very important role. In addition, it can serve as \na specific anchor protein for bacteria and viruses and \nis also involved in the regulation of redox reactions in \ncells[6-8].\nIt was found that estrogen in the female could \nsignificantly regulate the expression of ANXA2, \nwhich could affect the incidence of AM by means of \nendometrial metastasis and angiogenesis. In recent \nyears, it was reported that the EMT is the initiator of the \npathogenesis of AM [9]. Studies on in vitro AM model \nconfirmed that estrogen can significantly up-regulate \nANXA2 and induce the occurrence of EMT, suggesting \nthat ANXA2 may be related to the occurrence of AM. \nSome studies have also confirmed that ANXA2 plays \nan active role in AM development by endometrial \nmetastasis and angiogenesis. In this study, the \nexpression of ANXA2 in AM tissues and serum of \nthe eutopic endometrium and ectopic endometrium \nwas detected by immunohistochemical S-P assay \nand enzyme-linked immunosorbent assay (ELISA) \nto explore its mechanism of action in the occurrence \nand development of AM, so as to provide a reliable \ntheoretical basis for the clinical diagnosis and treatment \nof AM and the development and application of relevant \nmarkers.\nFrom April 2016 to October 2018, 22 AM patients, \naged 31-46 y, with an average age of 42.55±4.82 y, \nwho underwent laparoscopic hysterectomy in the \nHeilongjiang Provincial Hospital were enrolled as the \nobservation group. The eutopic and ectopic endometria \nwere used for experiments. Thirty two preoperative \npatients with synchronous uterine fibroids were \nselected, the patients were aged 33-47 y, with an average \nage of 43.64±5.65 y. All of them were confirmed to \nhave uterine fibroids and normal endometrium on \npostoperative pathology.\nDuring the same period, 22 cases of normal endometrium \nin patients with uterine fibroids undergoing laparoscopic \nhysterectomy in the Heilongjiang Provincial Hospital \nwere selected, who were aged from 32 to 46 y, with an \naverage age of 42.60±4.74 y as the control group. Thirty-\ntwo preoperative serums of patients with synchronous \nuterine fibroids were selected. The patients were 33 \nto 48 y, with an average of 44.02±5.95 y. All of them \nwere confirmed to have uterine fibroids and normal \nendometrium by postoperative pathology.\nAll specimens were strictly screened as premenopausal \nwomen with no endometrial atypical hyperplasia. The \npatient should have no other endocrine, immune and \nmetabolic diseases, malign ant tumors, and did not \nreceive any hormone or immunotherapy within 3 mo \nbefore surgery. This study was approved by the medical \nethics committee of the Heilongjiang Provincial \nHospital, all patients were informed and gave consent \nto the content of this study and all pathological sections \nwere reviewed by the pathologist.\nIn this study, the tissue was excised by paraffin \nembedding surgery and stained with a 4 μm thick \nsection. All specimens were operated according to kit \ninstructions under the same conditions, and PBS was \nused as negative control instead of primary antibody. \nThe specific operation process is shown in fig. 1.\nThe specific reaction of antigen and antibody is used \nto connect the substance to enzyme and then the color \nreaction is produced between enzyme and substrate for \nquantitative determination. The subject to be measured \nmay be an antibody or an antigen. The method of labeling \nwith fluorescein is called immunofluorescence and the \ncommonly used luciferin is fluorescein isothiocyanate, \nrhodamine and so on. The commonly used enzyme is \nhorseradish peroxidase. The enzyme reacts with the \nsubstrate to form an opaque deposit, which shows the \nposition of the presence of the antigen. In this study, \n3 ml of venous blood was taken on an empty stomach \nin the morning before the open hysterectomy and the \nserum was separated and stored at –80°. The enzyme \nlabeling instrument was Bio-Rad680 product (Bio-Rad, \n\nwww.ijpsonline.com\nSpecial Issue 2, 2020 Indian Journal of Pharmaceutical Sciences\n37\nUSA). The procedure described in the kit was followed \nstrictly during testing.\nThe data were processed by SPSS20.0 statistical \nsoftware and the measurement data were expressed as \nmean±standard deviation. t test was used to compare \nserum concentrations between groups, and p<0.05 was \nconsidered statistically significant.\nStudies have found that the promotion of endometrial \ntissue growth, distant metastasis and angiogenesis \nin AM patients is achieved through the mechanism \nof ANXA2-induced EMT. The basic structure of \nANXA2 contains 339 amino acids, consisting of a \n3kD N-terminus and a 33kD C-terminal domain. As \na calcium-binding cytoskeleton protein, it is involved \nin angiogenesis, proliferation, apoptosis, transduction \nof calcium signal and cell growth regulation. The \nmechanism of its induction of EMT in AM may be that \nit is hydrolyzed into fibrinolytic enzyme after binding \nto fibrinolytic progenitor and fibrinolytic enzyme has \nthe function of degrading extracellular matrix and \nbasement membrane of peripheral blood vessels. It \ncan also bind to adhesion molecules on the surface of \ncell, so as to change the adhesion between cells and \nbetween cells and matrix, enhance the antiapoptotic \nability and motor ability of cells and develop EMT. The \nlevels of ANXA2 in the endometrial tissues of the two \ngroups were compared. It was found that the level of \nANXA2 in the normal endometrial tissue of the control \ngroup was significantly lower than that in the ectopic \nand the eutopic endometrium tissue in the observation \ngroup (p<0.05) and the level of ANXA2 in the ectopic \nendometrial tissue was significantly higher than that in \nthe eutopic endometrial tissue (p<0.05), as shown in \nTable 1 and fig. 2.\nANXA2 is a calcium-mediated phospholipid-binding \nprotein, a member of the membrane protein family, \nwidely distributed in the nucleus, cytoplasm and \ncytoplasmic membrane surface. In cells, ANXA2 is \ninvolved in a series of important life processes including \nmembrane formation, membrane transport, endocytosis, \nexocytosis, cell proliferation, signal transduction, \ndifferentiation and apoptosis. In patients with elevated \nAM, estrogen might increase cyclooxygenase 2 by up-\nregulating ANXA2, thereby increasing the production \nof prostaglandin E2 and promoting the production and \nexacerbation of dysmenorrhea. EMT is also closely \nrelated to the high estrogen environment, and estrogen-\ninduced EMT is one of the important mechanisms of \nAM development, in which ANXA2 may play a key \nrole. In this study, the ANXA2 level in serum in the \nobservation group was significantly higher than that in \nthe control group, and the difference between the two \ngroups was statistically significant (p<0.05), as shown \nin Table 2. This suggested that the level of ANXA2 in \nserum was also significantly increased in patients with \nAM, which may be related to the fact that the level of \nANXA2 was up-regulated by the disorder of estrogen \nmetabolism in patients.\nConventional xylene \ndewaxing and gradient \nalcohol dehydration; xylene \nⅠ 20 min, xylene Ⅱ20 min, \n100% alcohol 10 min, 100% \nalcohol 10 min, 95% alcohol \n5 min, 80% alcohol 5 min, \n70% alcohol 5 min\nBake slices at 68° for 20 minutes\nDAB/H2O2 reactive dyeing and full \nrinsing of tap water; Hematoxylin re-\ndyeing, conventional dehydration, \ntransparency, drying, sealing\nBlocking the inactivation of \nendogenous peroxidase: \nincubating at 3% H2O2 37°  \nfor 10 minutes, rinsing with \nPBS for 3*5 minutes\nAntigen repair: boil in \n0.01M citric acid buffer \n(PH6.0) for 15 - 20 minutes\n（95°）, then rinse the \ncylinder with cold water to \naccelerate cooling to room \ntemperature, and rinse with \nPBS for 3*5 minutes\nNormal sheep serum \nworking fluid was sealed at \n37° for 10 minutes, and \ndumped without washing.\nDrip streptomycin-\novalbumin working solution \nlabeled with horseradish \nperoxidase, incubate at 37 °\nfor 30 minutes, and rinse \nwith PBS for 3*5 minutes\nDrop the first antibody and \nincubate overnight in \nrefrigerator at 4 °. and rinse \nthe PBS for 3*5 min (PBS \nbuffer instead of the first \nantibody as negative \ncontrol); \nThe second antibody was \nlabeled with biotin, \nincubated at 37° for 30 \nminutes and washed with \nPBS for 3*5 minutes.\nFig. 1: S-P staining flow chart\n\nwww.ijpsonline.com\nSpecial Issue 2, 2020Indian Journal of Pharmaceutical Sciences\n38\nANXA2 was found to be abnormally expressed in \ncervical cancer, ovarian cancer, choriocarcinoma \nand other gynecological malignancies. AM also had \nsimilar biological behavior to malignant tumors. \nIt was speculated that ANXA2 may be abnormally \nexpressed in human AM and promote the occurrence \nand development of AM, but relevant studies have not \nbeen reported. The level of ANXA2 in peripheral blood \nof patients with uterine fibroids was significantly lower \nthan that of patients with AM (p<0.05). This indicated \nthat the level of ANXA2 in peripheral blood had a \ncertain differentiation effect between AM and uterine \nfibroids, suggesting that ANXA2 may be a new marker \nfor AM diagnosis. Therefore, changes in the level of \nANXA2 need to be monitored to assist in the diagnosis \nand treatment of patients with AM.\nAM mostly occurs in middle-aged women and its \nmain clinical symptoms are secondary dysmenorrhea \nand increased menstrual volume, as well as infertility. \nCurrently, hysterectomy is the most effective treatment \noption. However, this surgical method is often hard \nto accept for some patients who have fertility needs \nor desire to retain the uterus and the most accurate \nmeans of diagnosis nowadays is postoperative \nhistopathological examination. Therefore, it is of great \nsignificance to search for an early biomarker to evaluate \nAM. It has been reported that the incidence of AM \nmay be related to EMT and ANXA2 could induce this \ntransformation mechanism. ANXA2, as the co-receptor \nof tissue plasminogen activator and plasminogen, can \nbe produced by plasminogen mediated and catalyzed by \ncell surface layer. In endothelial cells, mononuclear or \nmacrophages, and some tumor cells, it can participate \nin biological membrane and ion channels, cytoskeleton, \ncell proliferation, differentiatio n, and other processes, \nwhich are associated with endometrial lesions.\nThe results of this study showed that the level of ANXA2 \nin the normal endometrial tissue was significantly lower \nthan that in the eutopic and ectopic endometria (p<0.05), \nsuggesting that the level of ANXA2 in the endometrial \ntissue of patients with AM was significantly increased. \nThis could be related to the involvement of ANXA2 in \nthe pathogenesis and progression of AM. As a calcium-\nbinding cytoskeletal protein, ANXA2 can regulate the \ngrowth, proliferation, and apoptosis of cells or blood \nGroup Cases ANXA2\nControl group Normal endometrium 22 3.80±2.62\nObservation \ngroup\nEutopic endometrium 22 7.88±2.04\nEctopic endometrium 22 8.42±1.98\nTABLE 1: COMPARISON OF ANXA2 LEVELS IN \nENDOMETRIAL TISSUE BETWEEN THE TWO \nGROUPS\nMean±standard deviation, ng/ml\n \n(a)  \n \n \n(b)  \n \n \n(c)  \nFig. 2: Expression of ANXA2 in the intimal tissue of two groups \nof patients\n(a) Expression of ANXA2 in ectopic endometrium of patients \nwith adenomyosis. (b) Expression of ANXA2 in the eutopic \nendometrium of patients with adenomyosis. (c) Expression of \nANXA2 in normal endometrium\nGroup Cases ANXA2\nControl group 22 7.78±2.11\nObservation group 22 8.28±1.72\nt - 2.562\nP - ˂0.05\nAdenomyosis group 32 3.14±0.52\nFibroid group 32 1.04±0.28\nt - 20.774\nP - ˂0. 05\nTABLE 2: COMPARISON OF ANXA2 LEVELS IN \nSERUM OF TWO GROUPS OF PATIENTS\nMean±standard deviation, ng/ml\n\nwww.ijpsonline.com\nSpecial Issue 2, 2020 Indian Journal of Pharmaceutical Sciences\n39\nkappa B alpha are regulated by estradiol in endometrial cells. \nTurk J Obstet Gynecol 2018;15(1):50-9.\n4. Zou Y , Liu FY , Wang LQ. Downregulation of DNA \nmethyltransferase 3 alpha promotes cell proliferation and \ninvasion of ectopic endometrial stromal cells in adenomyosis. \nGene 2017;604:41-7.\n5. Lin X, Dai Y , Xu W. Hypoxia Promotes Ectopic Adhesion \nAbility of Endometrial Stromal Cells via TGF-β1/Smad \nSignalling in Endometriosis. J Endocrinol 2018;159(4):1630-\n41.\n6. Ji F, Yang X, He Y . Aberrant endometrial DNA methylome \nof homeobox A10 and catechol-O-methyltransferase in \nendometriosis. J Assist Reprod Genet 2017;34(3):409-15.\n7. Patel BG, Rudnicki M, Yu J, Shu Y , Taylor RN. Progesterone \nresistance in endometriosis: origins, consequences and \ninterventions. Acta Obstet Gynecol Scand 2017;96(6):623-32.\n8. Zhao L, Gu C, Ye M, Zhang Z, Li L, Fan W, et al. Integration \nanalysis of microRNA and mRNA paired expression profiling \nidentifies deregulated microRNA-transcription factor-gene \nregulatory networks in ovarian endometriosis. Reprod Biol \nEndocrinol 2018;16(1):4.\n9. Guo SW. Fibrogenesis resulting from cyclic bleeding: the \nHoly Grail of the natural history of ectopic endometrium. Hum \nReprod 2018;33(3):353-6.\nvessels and the conduction of calcium signals and can \nbe hydrolyzed into fibrinolytic enzyme by combining \nwith plasminogen, resulting in the degradation of \nextracellular matrix and vascular basement membrane. \nIn addition, it can be combined with adhesion \nmolecules on the cell surface, leading to changes in \nadhesion between cells and the matrix, strengthening \nthe antiapoptosis and motor ability of cells themselves, \nand eventually causing AM.\nIn this study, for the first time, ANXA2 in serum of \npatients with AM was determined using ELISA before \nsurgery and it was significantly higher than that in \npatients with uterine fibroids, suggesting that ANXA2 \ncould serve as a new marker for AM diagnosis. \nTherefore, changes in the level of ANXA2 should be \nmonitored clinically to assist the diagnosis and treatment \nof patients with AM, so as to grasp their conditions and \npredict prognosis changes more accurately. However, \nthis study needs to be verified by multi-center samples.\nREFERENCES\n1. Liu LX, Wu YG, Zheng J. Increased annexin A2 and decreased \nβ-catenin in adenomyosis contribute to adenomyosis-\nassociated dysmenorrhea. Histol Histopathol 2017;32:11906.\n2. Jiang C, Liu C, Guo J. The Expression of Toll-like receptors \nin eutopic and ectopic endometrium and its implication in \nthe inflammatory pathogenesis of adenomyosis. Sci Rep \n2017;7(1):7365.\n3. Arlıer S, Arıcı A. Tumor necrosis factor alfa and interleukin \n1 alfa induced phosphorylation and degradation of inhibitory \nThis article was originally published in a special issue:  \nSpecial issue on “Drug Development and Human Health in \nChina” \nIndian J Pharm Sci 2020:82(1)spl issue2;35-39\nThis is an open access article distributed under the terms of the Creative \nCommons Attribution-NonCommercial-ShareAlike 3.0 License, which  \nallows others to remix, tweak, and build upon the work non-commercially,  \nas long as the author is credited and the new creations are licensed under \nthe identical terms","source_license":"CC0","license_restricted":false}