{"paper_id":"c10ab5cb-545d-46c4-95e3-7ada49205d44","body_text":"The Apoptotic, Angiogenic and Cell Proliferation\nGenes CD63, S100A6 e GNB2L1 are Altered in\nPatients with Endometriosis\nOs genes apoptóticos, angiogênicos e de proliferação\ncelular CD63, S100A6 e GNB2L1 estão alterados em\npacientes com endometriose\nValéria Aguiar Gomes 1 Camila de Moraes Bonocher 1 Júlio César Rosa-e-Silva 1\nCláudia Cristina Paro de Paz 2 Rui Alberto Ferriani 1 Juliana Meola 1\n1 Department of Gynecology and Obstetrics, School of Medicine of\nRibeirao Preto, Universidade de São Paulo, Ribeirão Preto, SP , Brazil\n2 Department of Genetics, School of Medicine of Ribeirao Preto,\nUniversidade de São Paulo, Ribeirão Preto, SP, Brazil\nRev Bras Ginecol Obstet 2018;40:606 –613.\nAddress for correspondence Juliana Meola, PhD, Departamento de\nGinecologia e Obstetrícia, Escola de Medicina de Ribeirão Preto,\nUniversidade de São Paulo, Av. Bandeirantes, 3900, HC/FMRP,\n14049900, Monte Alegre, Ribeirao Preto, SP, Brazil\n(e-mail: jumeola@yahoo.com.br).\nKeywords\n► endometriosis\n► gene expression\n► molecular genetics\n► pathophysiology\n► real-time PCR\nAbstract Objective The aim of the present study was to analyze the expression of the CD63,\nS100A6,a n d GNB2L1genes, which participate in mechanisms related to the complex\npathophysiology of endometriosis.\nMethods A case-control study was conducted with 40 women who were diagnosed with\nendometriosis, and 15 fertile and healthy women. Paired samples of eutopic endometrium\nand endometriotic lesions (peritoneal and ovarian endometriotic implants) were obtained\nfrom the women with endometriosis in the proliferative ( n ¼ 20) or secretory phases\n(n ¼ 20) of the menstrual cycle. As controls, paired endometrial biopsy samples were\ncollected from the healthy women in the proliferative ( n ¼ 15) and secretory ( n ¼ 15)\nphases of the same menstrual cycle. We analyzed the expression levels of theCD63, S100A6,\nand GNB2L1 genes by real-time polymerase chain reaction.\nResults An increase in CD63, S100A6,a n dGNB2L1 gene transcript levels was observed\nin the ectopic implants compared with the eutopic endometrium of the women with\nand without endometriosis, regardless of the phase of the menstrual cycle.\nConclusion These findings suggest that the CD63, S100A6 ,a n dGNB2L1 genes may be\ninvolved in the pathogenesis of endometriosis, since they participate in mechanisms\nsuch as inhibition of apoptosis, angiogenesis and cell proliferation, which lead to the\nloss of cell homeostasis in the ectopic endometrium, thus contributing to the\nimplantation and survival of the tissue in the extrauterine environment.\nResumo Objetivo O objetivo do presente estudo foi analisar a expressão dos genes CD63,\nS100A6 e GNB2L1, que participam em mecanismos relacionados à complexa fisiopa-\ntologia da endometriose.\nreceived\nFebruary 3, 2018\naccepted\nJune 21, 2018\nDOI https://doi.org/\n10.1055/s-0038-1673364.\nISSN 0100-7203.\nCopyright © 2018 by Thieme Revinter\nPublicações Ltda, Rio de Janeiro, Brazil\nOriginal Article\nTHIEME\n606\n\n\nIntroduction\nEndometriosis is a benign gynecological disease that affects at\nleast 10% of women of reproductive age. 1 It is de fined by the\ngrowth of endometrial tissue outside the uterine cavity, which\nis known as ectopic tissue. Although endometriosis is consid-\nered a benign disorder, there are some similarities with cancer;\nboth share invasive potential for the implantation and main-\ntenance of ectopic tissue. Altered immune response, adhesion,\ninvasion, cell proliferation, inhibition of apoptosis, angiogene-\nsis, local estrogen production, and response to injury are\nimportant events in the pathogenesis of endometriosis.\n2\nRecent studies have demonstrated the molecular differ-\nences between the eutopic and the ectopic endometrium,\nsuggesting that distinct patterns of gene expression are\ninvolved in the development of endometriosis.\n3–10 The alter-\nations in the expression of certain key genes at speci fic\nmoments can determine normal and abnormal physiological\nprocesses and dysregulate the relevant metabolic pathways\ninfluencing the formation of lesions. Therefore, genetic\nstudies are necessary to better understand and de fine the\nmolecular etiology of this disease.\nPrevious studies have shown the dysregulation of some\ngenes in endometriosis. These genes are also involved in\nseveral pathways, such as the inhibition of apoptosis, cell\nsurvival, angiogenesis, and cell proliferation, which suggests\ntheir participation in the pathophysiology of endometriosis.\n3,4\nIn addition, our previous study demonstrated the dysregula-\ntion of the CD63, GNB2L,a n d S100A6 genes in endometriosis.\nTheir involvement in the aforementioned processes and the\ncorrelation with cancer suggests that the expression of these\ngenes possibly leads to the establishment and survival of\nendometriotic implants.\n3,4 Therefore, the evaluation of these\ngenes can clarify some of the mechanisms that underlie the\ncomplex pathophysiology of endometriosis.\nThe purpose of the present study was to compare the\nexpression levels of the CD63, GNB2L1 ,a n d S100A6 genes in\nthe endometrial tissue from women without endometriosis,\nin the eutopic and the ectopic endometrium (pelvic and\novarian endometriotic implants), in the proliferative and\nsecretory phases of the menstrual cycle from the patients\nwith endometriosis.\nMethods\nEthics, Setting, and Duration\nA case-control study was conducted on patients with and\nwithout endometriosis in the proliferative and secretory\nphases of the menstrual cycle. The present study was\np e r f o r m e df r o m2 0 1 0t o2 0 1 2a tt h eS e c t o ro fH u m a n\nReproduction from the Department of Gynecology and\nObstetrics of Faculdade de Medicina de Ribeirão Preto da\nUniversidade de São Paulo (FMRP-USP), in the state of São\nPaulo, Brazil. The present study was approved by the\nInstitutional Review Board of the FMRP-USP, protocol\nHCRP n\no 11736/2004, and is linked to a bank of endome-\ntriosis tissues, which is also approved, protocol HCRP n o\n9699/2006. All subjects (those with endometriosis and the\ncontrols) signed a written informed consent to participate\nin the study. The present work was performed in accor-\ndance with the ethical standards of the Declaration of\nHelsinki.\nParticipants and Eligibility Criteria\nThe women were included if they were 18 to 40 years old, had a\nbody mass index (BMI)< 30 kg/m2, were not menopausal, had\nnot been taking any hormonal therapy for at least 3 months\nbefore sample collection, had no reproductive disorder or\ntumor, and had a regular menstrual cycle. Other exclusion\ncriteria included smoking, alcoholism, recreational drug use,\nMétodos Um estudo caso-controle foi realizado com 40 mulheres diagnosticadas\ncom endometriose e 15 mulheres férteis e saudáveis. Amostras pareadas de endomé-\ntrio eutópico e de lesões endometrióticas (implantes endometrióticos peritoneais e\novarianos) foram obtidas de mulheres com endometriose nas fases proliferativa\n(n ¼ 20) ou secretora ( n ¼ 20) do ciclo menstrual. Como controle, amostras pareadas\nde biópsia endometrial foram coletadas de mulheres saudáveis nas fases proliferativa\n(n ¼ 15) e secretora ( n ¼ 15) no mesmo ciclo menstrual. Foram analisados os níveis de\nexpressão dos genes CD63, S100A6 e GNB2L1 por reação em cadeia da polimerase em\ntempo real.\nResultados Foi observado um aumento nos níveis de transcritos dos genes CD63,\nS100A6 e GNB2L1 em implantes ectópicos quando comparado ao endométrio eutópico\nde mulheres com e sem endometriose, independente da fase do ciclo menstrual.\nConclusão Estes achados sugerem que os genes CD63, S100A6 e GNB2L1 podem estar\nenvolvidos na patogênese da endometriose, pois participam de mecanismos como\ninibição de apoptose, angiogênese e proliferação celular, os quais levam à perda da\nhomeostase celular no endométrio ectópico e, portanto, contribuem para o implante e\na sobrevivência do tecido no ambiente extrauterino.\nPalavras-chave\n► endometriose\n► expressão gênica\n► genética molecular\n► fisiopatologia\n► PCR em tempo real\nRev Bras Ginecol Obstet Vol. 40 No. 10/2018\nThe Apoptotic, Angiogenic and Cell Proliferation Genes cd63, s100a6 ,a n d gnb2l1 Gomes et al. 607\n\n\nany systemic disease (systemic arterial hypertension, diabetes\nmellitus, immune system diseases, or thyroid diseases).\nThe patients of the endometriosis group were diagnosed\nthrough laparoscopy and histopathological analyses. An\nexperienced surgeon performed the laparoscopy, and the\nstage of endometriosis was determined according to the\nclassification of the American Society for Reproductive Med-\nicine (1997).\n11\nThe control group consisted of women of reproductive age\n(18 to 40 years old), who were submitted to laparoscopy for\ntubal ligation. These women did not have endometriosis,\nfibrosis, pelvic adhesion, or infertility.\nSample Collection and Processing\nTissue Samples\nThe paired tissue samples of the eutopic endometrium and of\nthe endometriotic lesions (peritoneal or ovarian lesions)\nwere collected from 40 women with endometriosis during\nlaparoscopy, and the eutopic endometrial samples were\ncollected using a Euro-Med Novak Endometrial Curette\n(CooperSurgical, Inc., Trumbull, CT, USA). Only one sample\nwas collected per patient. In the control group, two paired\nbiopsy tissue samples were collected from 15 healthy wom-\nen according to the phase of the menstrual cycle. The phases\nof the cycle were determined by histological dating. The\nsamples were stored frozen at - 80° C for later analysis after\ntreatment with RNA later Stabilization Solution (Thermo\nFischer Scienti fic, Waltham, MA, USA).\nTotal RNA Extraction\nThe samples were washed with phosphate-buffered saline\n(PBS) solution (1x) (8.50 g/L NaCl, 1.11 g/L Na2HPO4,a n d2 . 8 1\ng/L Na2HPO4,12H2O, 0.20 g/L, and KH 2PO4, pH 7.0) to remove\nthe RNA later solution from the tissues. Next, total RNA\n(50 mg tissue) was extracted using the TRIzol reagent\n(Thermo Fischer Scienti fic, Waltham, MA, USA) according\nto the manufacturer’ s instructions. After the treatment of the\nsamples with DNase I (Thermo Fischer Scienti fic, Waltham,\nMA, USA), RNA integrity was con firmed by the presence of\nthe ribosomal bands (28S and 18S) after analysis by 1%\nagarose gel electrophoresis. Total RNA concentrations were\ndetermined using a NanoDrop 2000c spectrophotometer\n(Thermo Fischer Scienti fic, Waltham, MA, USA) at 260 nm.\nThe RNA was stored at - 80° C until further processing.\nRelative Quanti fic a t i o nb yR e a l - t i m eP o l y m e r a s eC h a i n\nReaction\nAn aliquot (1 µg) of total RNA from each sample was reverse-\ntranscribed using random primers from the High Capacity\ncDNA Archive kit (Thermo Fischer Scienti fic, Waltham, MA,\nUSA) according to the manufacturer ’ s instructions. The reac -\ntion was performed in a Piko Thermal Cycler (Thermo Fischer\nScientific, Waltham, MA, USA) for 10 minutes at 25° C; 2 hours\nat 37° C; 5 minutes at 85 °C, and 5 minutes at 4° C.\nThe relative quanti fication (RQ) of the expression of the\nselected genes in the collected samples was performed using an\nABI PRISM 7500 FAST equipment (Applied Biosystems, Foster\nCity, CA, USA). The reactions were performed using the TaqMan\nGene Expression Assay system, being the TaqMan probe with\nFAM dye label on the 5 ′ end and minor groove binder (MGB)\nand nonfluorescent quencher (NFQ) on the 3 ′ end. This infor-\nmation is an explanation of which TaqMan Gene Expressed\nAssay was used. (Applied Biosystems, Foster City, CA, USA). The\nassay IDs of the probes used were CD63 (Hs00156390_m1),\nGNB2L1(Hs00272002_m1),S100A6(Hs00170953_m1),GAPDH\n(Hs99999905_m1), and ACTB (Hs99999903_m1).\nReal-time polymerase chain reaction (It ’ s qRT-PCR. Real-\nTime Quantitative Reverse Transcription PCR. RT-PCR is\nReverse transcription-polymerase chain reaction) was per-\nformed in triplicate for each sample using a reaction mixture\nwith a final volume of 20 µL consisting of the following:\n10 µL TaqMan Universal PCR Master Mix (2x) (Applied Bio-\nsystems, Foster City, CA, USA), 1 µL TaqMan Gene Expression\nAssay Mix (20x) (Applied Biosystems, Foster City, CA, USA),\nand 9 µL cDNA diluted 1:50. The reaction conditions were\n50° C for 2 minutes, 95° C for10 minutes, 40 cycles of 95° C for\n15 seconds and 60° C for 1 minute.\nA cDNA pool of the endometrial samples ( n ¼ 30)\nobtained from the control group was used as a reference\nsample (calibrator). The reference genes GAPDH and ACTB\nwere used for normalizing the reactions. Relative quanti fi-\ncation of the analyzed genes was calculated for each sample\n(control group ¼ 30; and endometriosis group ¼ 40) by the\n2\n-ΔΔCT method.12\nStatistical Methods\nStatistical analyses were performed with the aid of the SAS\n2003 software (SAS Institute Inc., Cary, NC, USA). The RQ values\nof the genes were log-transformed (log 10 [2-ΔΔCTt þ10]).\nLogarithmic transformation was necessary, since one of the\nassumptions (linearity) of the linear model analysis was not\nsatisfied.\nThe analysis of variance (ANOVA) was performed by\nincluding the effects of phases of the menstrual cycle (pro-\nliferative and secretory), the type of tissue (peritoneal lesion,\nendometrioma, eutopic endometrium of patients with and\nwithout endometriosis) as well as the interactions among\nthem in the statistical model.\nWe used the t-test for paired samples (eutopic endome-\ntrium versus ectopic lesions), and the Welch ANOVA test for\nunpaired samples (control endometrium versus eutopic\nendometrium of patients with endometriosis, control endo-\nmetrium versus lesions of patients with endometriosis, and\nperitoneal lesions versus ovarian lesions).\nData, which are log\n10 (2-ΔΔCT þ10)-transformed, are illus-\ntrated in ►Fig. 1 with mean and standard deviation (SD). The\nanalyses were satisfactory, with a test power (1- β) of at least\n80%, and the results were considered statistically signi ficant\nwhen p < 0.05.\nResults\nThere was no signi ficant difference between the endometri-\nosis and control groups regarding mean age and standard\ndeviation (33.87 /C6 2.69 and 33.5 /C6 4.37 years old,\nRev Bras Ginecol Obstet Vol. 40 No. 10/2018\nThe Apoptotic, Angiogenic and Cell Proliferation Genes cd63, s100a6 ,a n d gnb2l1 Gomes et al.608\n\n\nFig. 1 Relative expression of the CD63, GNB2L1 and S100A6 genes in human endometrial tissue throughout the menstrual cycle. ( A)R e l a t i v e\nexpression of the CD63 gene in the proliferative phase, ( B) relative expression of the CD63 gene in the secretory phase, ( C) relative expression of\nthe GNB2L1 gene in the proliferative phase, ( D)r e l a t i v ee x p r e s s i o no ft h eGNB2L1 gene in the secretory phase, ( E) relative expression of the\nS100A6 gene in the proliferative phase and ( F) relative expression of the S100A6 gene in the secretory phase. Control ¼ endometrium of women\nwithout endometriosis; Eutopic ¼ endometrium of women with endometriosis; Lesions ¼ endometriotic lesions (peritoneal and ovarian\nlesions). /C3 p < 0.05 versus control group (unpaired analyses). # p < 0.05 versus eutopic group (paired analyses). Data are shown as mean /C6\nstandard deviation.\nRev Bras Ginecol Obstet Vol. 40 No. 10/2018\nThe Apoptotic, Angiogenic and Cell Proliferation Genes cd63, s100a6 ,a n d gnb2l1 Gomes et al. 609\n\n\nrespectively). The control group consisted of 15 women of\nreproductive age. Fifteen biopsy samples were obtained\nduring the proliferative phase, and 15 during the secretory\nphase of the menstrual cycle. The endometriosis group\nconsisted of 40 women, and of these patients, 21 were\nattended to due to infertility, and 19 due to pelvic pain at\nthe outpatient clinics of marital infertility, and pelvic pain\nand endoscopy of the university hospital of the FMRP-USP.\nThe number of peritoneal lesions studied in the patients\nclassified as stage I ( n ¼ 6; in the secretory phase), stage II\n(n ¼ 11; 7 in the proliferative phase and 4 in the secretory\nphase), stage III ( n ¼ 2; in the proliferative phase), and stage\nIV (n ¼ 1; in the proliferative phase) was 20, and that of the\novarian endometrioma lesions studied in the patients classi-\nfied as stage III ( n ¼ 8; 4 in the proliferative phase and 4 in\nthe secretory phase) and stage IV ( n ¼ 12; 6 in the prolifer-\native phase and 6 in the secretory phase) was 20.\nThe results of gene expression in the analyzed tissues are\nillustrated in\n►Fig. 1 . There was no difference in the expres-\nsion of target genes between the peritoneal and ovarian\nlesions (data not shown). Thus, the peritoneal and endome-\ntrioma lesions were pooled for the remaining analyses.\nIncreased expression of the CD63, GNB2L1, and S100A6\ngenes was found in the endometriotic lesions regardless of\nthe phases of the menstrual cycle that were studied (\n►Fig. 1 ).\nFurthermore, increased expression of the CD63, and of the\nS100A6 genes (in the proliferative phase), and of the GNB2L1\ngene (in the secretory phase) was detected in the eutopic\nendometrium of the patients of the endometriosis group\ncompared with the control endometrium (\n►Figs. 1A , E\nand D; respectively). However, when the proliferative and\nsecretory phases were compared with each other in the\nendometrial control ( CD63 p ¼ 0.52; GNBL1 p ¼ 0.79;\nS100A6 p ¼ 0.052), eutopic endometrium ( CD63 p ¼ 0.38;\nGNBL1 p ¼ 0.41; S100A6 p ¼ 0.44) and endometriotic lesions\n(CD63 p ¼ 0.94; GNBL1 p ¼ 0.49; S100A6 p ¼ 0.24), no differ-\nence was observed.\nDiscussion\nThe present study identi fied the increased expression of the\nCD63, GNB2L1,a n d S100A6 genes in the ectopic lesions of\nwomen with endometriosis compared with the endometrium\nof the control group during both the proliferative and secretory\nphases of the menstrual cycle. In addition, the expression of\nthese genes had increased in the lesions compared with the\neutopic endometrium of the same endometriosis patient\nduring the secretory phase of the menstrual cycle. However,\nwhen the eutopic endometrium of the women with endome-\ntriosis were compared with the endometrium of the control\nwomen, the expression of the CD63 and of the S100A6 genes\nhad increased only in the proliferative phase in the endome-\ntrium of the women with endometriosis, and the increased\nexpression of the GNB2L1 gene was observed only in the\nsecretory phase.\nThese results are in agreement with our previously\nobtained results, which showed the increased expression of\nthe target genes in endometriotic lesions in a large-scale\nhybridization study based on subtractive libraries.\n3 However,\nin the present study, we used RT-PCR to quantify and confirm\nthe dysregulated expression of these genes during the phases\nof the menstrual cycle. This technique has been defined as the\ngold standard for transcript quanti fication due to its high\nsensitivity and good reproducibility. In addition, possible\nfalse-positive results can be revealed when this technique is\nused for the validation of screening methodologies. 13 The\nobtained results confirmed the increased expression of these\ngenes in endometriotic lesions.\nOne of the possible explanations for the differences\nobserved between the eutopic and the ectopic endometrium\nof the patients with endometriosis is that endometriosis is a\nmultifactorial disorder, which is in fluenced by genetic and\nenvironmental factors. Studies have shown that different\nendocrine environments, such as the peritoneal fluid and the\nintraovarian microenvironment of the lesions, are critical to\nendometrial implantation, suggesting that the interaction\nbetween these different endocrine environments and the\nectopic endometrium is important in the pathophysiology of\nendometriosis.\n14\nIn the present study, we have detected the increased expres-\nsion of theCD63 gene in endometrial lesions compared with the\neutopic tissue of women with and without endometriosis. The\nhuman CD63 gene, which codes for a tetraspanin, has been\nmapped to the chromosome region 12q13, and was first\ndiscovered as a surface antigen that is abundantly expressed\nin cells in the initial stage of melanoma and on the surface of\nactivated blood platelets.15 The CD63 gene interacts directly\nand indirectly with various molecules, such as integrins, other\ntetraspanins, cell surface receptors, kinases, protein adaptors,\nand other proteins, including the L6 antigen, syntenin-1, TIMP-\n1, and MT1-MMP.\n16–27 The CD63 gene was recently identified to\nbind to the TIMP-1 protein, which is known to be involved in\nseveral cellular processes and in tumor development. 26 The\ncomplex of CD63,T I M P - 1a n dβ1 integrin mediates the activa-\ntion of cell survival pathways through the activation of the focal\nadhesion kinase (FAK), as well as of the PI3-K and extracellular\nsignal– regulated kinase (ERK) pathways, and to the inhibition\nof apoptosis.\n26,28,29 Therefore, we hypothesized that the in-\ncrease in the expression of theCD63 gene may be related to the\npromotion of the survival of ectopic endometrial cells, thus\nfavoring the development of endometriotic lesions.\nAnother important gene that was suggested by Meola et al3\nand Dentillo et al 4 to be involved in the pathophysiology of\nendometriosis is GNB2L1, which is commonly called RACK1\n(receptor for activated C -kinase 1) and is located in the chro-\nmosome region 5q35.3. It possesses 7 WD domains (trypto-\nphan-aspartate), which provides RACK1 with the potential to\nact as an adaptor or scaffold protein in the interactions with\nvarious molecules.3,4,30 Many cellular functions are attributed\nto RACK1, such as cell growth, chemotactic adhesion and\nmigration, which are mediated by the interaction with integrin\nand Src, as well as the suppression of apoptosis, anti-in flam-\nmation and angiogenesis.31–35\nIn our study, the expression of theGNB2L1 gene was consid-\nerably higher in endometriotic lesions than in the eutopic\nendometria of women with and without endometriosis. Studies\nRev Bras Ginecol Obstet Vol. 40 No. 10/2018\nThe Apoptotic, Angiogenic and Cell Proliferation Genes cd63, s100a6 ,a n d gnb2l1 Gomes et al.610\n\n\nhave indicated that the overexpression of this gene positively\nregulates cell adhesion and migration.36 The increased expres-\nsion of the GNB2L1gene and its interaction with IGF-IR aug-\nmented the mobility of transformed MCF-7 cells, showing that\nthis gene promotes the anchorage-independent growth of\novarian tumor cells via the insulin growth factor 1R (IGF-1R)/\nSTAT3 pathway, whereas other authors had reported discordant\nresults when analyzing the Src pathway.\n37–40 Moreover, the\nGNB2L1 gene is also overexpressed during angiogenesis, and in\nbreast and colon tumors, non-small cell lung carcinoma, oral\nsquamous cell carcinoma, and melanoma. 31,41–44 Thus, the\nupregulation of the expression of the GNB2L1 gene suggests\nits participation in processes such as angiogenesis, suppression\nof apoptosis, and cell proliferation, which are the steps involved\nin the pathology of endometriosis.\nThe third gene evaluated was S100A6, whose expression\nhad increased considerably in endometriotic lesions compared\nwith the eutopic endometrium of women with and without\nendometriosis. TheS100A6 gene, whose protein product is also\nknown as calcyclin, has Ca\n2þ-binding sites. 45 Some of the\nfunctions associated with this protein and changes in the\nexpression of this gene include cytoskeleton dynamics, cell\ncycle, apoptosis, overexpression in cells with proliferative\npotential, and increased induction of cell proliferation.\n46–51\nLiu et al 52 showed that the overexpression of the S100A6\ngene in endometrial stromal cells promoted the upregulation\nof β-catenin expression. The β-catenin is an essential com-\nponent of the Wnt/ β-catenin signaling pathway, which has\nbeen demonstrated to be activated in the mid-secretory\nendometrium of infertile patients with endometriosis.\n53\nThis pathway is involved in the control of proliferation,\nmigration, and invasion, which are important steps in the\npathogenesis of endometriosis. Recently, Zhang et al\n54 dem-\nonstrated that inhibition of the expression of the S100A6\ngene signi ficantly reduced the migratory ability of eutopic\nendometrial stromal cells, induced their apoptosis, and had\nantiproliferative effect. In addition, both increased protein\nlevels and increased transcript levels of the S100A6 gene have\nbeen observed in many types of tumors.\n54\nThe increased protein levels of the S100A6 gene promote\ncell apoptosis under oxidative stress.55 However, the proapop-\ntotic function of this gene is contradictory, as reduced expres-\nsion of this gene has been observed during the apoptosis of\nhuman breast cancer cells, and the expression of the S100A6\ngene has also been demonstrated to inhibit the apoptosis of\ncardiac myocytes.\n56,57 An increased expression of this gene\nincreases cell adhesion and inhibition of cell invasion, and\npromotes mobility in a way that is yet to be understood. 58,59\nHowever, when its expression is insuf ficient or is negatively\nregulated, this gene inhibits the proliferation of fibroblasts,\nosteoblasts, and pancreatic tumor cells. 46,60,61 Since the\nS100A6 gene is involved in cell cycle progression, cell differen-\ntiation, the interactions with the cytoskeleton, and cell prolif-\neration, it is possible that the changes in the expression of this\ngene that were observed in the present study could be\nimplicated in the development of endometriosis.\nNo differences in the levels of expression of the studied genes\nwere observed when the proliferative and secretory phases\nwere compared with each other in the endometrial control, in\neutopic endometria and in endometriotic lesions. However, the\neutopic endometria of women with endometriosis had an\nincreased expression of the CD63 and S100A6 genes in the\nproliferative phase, while the increased expression of the\nGNB2L1 gene was observed in the secretory phase when com-\npared with the control group; these results could be explained\nby the changes related to the disease. Although some authors\nrelate the changes of gene expression with hormonal fluctua-\ntions, there are few studies and there are controversies in the\nliterature regarding the genes studied. Okada et al\n62 showed\nthat the mRNA levels of the CD63 gene were substantially\nreduced during the secretory phase of the menstrual cycle\ncompared with the proliferative phase, and that the expression\nof the mRNAs is negatively regulated by progesterone, thus\nassociating this gene with functions such as proliferation and\ndifferentiation of the endometrium (decidualization). In con-\ntrast, Brar et al\n63 found no change in the expression of this gene\nduring decidualization. Tong et al64 studied the expression of the\nS100A6 gene in the human endometrium throughout the\nmenstrual cycle, and no change was detected.\nA limitation of our study was the small sample evaluated\ndue to the restrictive eligibility criteria adopted, limiting the\ngeneralization of the study.\nConclusion\nOur results suggest that the altered expression of the CD63,\nS100A6,a n d GNB2L1 genes may be involved in the patho-\nphysiology of endometriosis. This is because the CD63,\nRACK1 and calcyclin proteins participate in the inhibition\nof apoptosis, angiogenesis and cell proliferation, which can\nlead to the loss of cell homeostasis in the ectopic endometri-\num. Future studies are needed to assess the function of the\nidentified genes, and to characterize their roles in the\ndevelopment of the disease. Another aspect that should be\naddressed is that our study was based on the premise that\ntissue invasion occurs due to the reflux of viable endometrial\ncells. Although this idea is still one of the most accepted\ntoday, it is only a hypothesis that has not been con firmed.\nConflict of Interests\nNone to declare.\nAcknowledgments\nWe are grateful to all the patients for their participation in\nthe present study and to the multidisciplinary team of the\nHuman Reproduction Division of the Department of\nGynecology and Obstetrics at FMRP-USP for the sample\ncollection and technical support.\nReferences\n1 Yang WC, Chen HW, Au HK, et al. Serum and endometrial markers.\nBest Pract Res Clin Obstet Gynaecol 2004;18(02):305 – 318 Doi:\n10.1016/j.bpobgyn.2004.03.003\n2 Nap AW, Groothuis PG, Demir AY, Evers JL, Dunselman GA.\nPathogenesis of endometriosis. Best Pract Res Clin Obstet Gynae-\ncol 2004;18(02):233 – 244 Doi: 10.1016/j.bpobgyn.2004.01.005\nRev Bras Ginecol Obstet Vol. 40 No. 10/2018\nThe Apoptotic, Angiogenic and Cell Proliferation Genes cd63, s100a6 ,a n d gnb2l1 Gomes et al. 611\n\n\n3 Meola J, Rosa e Silva JC, Dentillo DB, et al. Differentially expressed\ngenes in eutopic and ectopic endometrium of women with endo-\nmetriosis. Fertil Steril 2010;93(06):1750 – 1773 Doi: 10.1016/j.\nfertnstert.2008.12.058\n4 Dentillo DB, Meola J, Rosa e Silva JC, et al. Deregulation of LOXL1\nand HTRA1 gene expression in endometriosis. Reprod Sci 2010;17\n(11):1016– 1023 Doi: 10.1177/1933719110377662\n5 Honda H, Barrueto FF, Gogusev J, Im DD, Morin PJ. Serial analysis\nof gene expression reveals differential expression between endo-\nmetriosis and normal endometrium. Possible roles for AXL and\nSHC1 in the pathogenesis of endometriosis. Reprod Biol Endocri-\nnol 2008;6:59 Doi: 10.1186/1477-7827-6-59\n6 Filigheddu N, Gregnanin I, Porporato PE, et al. Differential expres-\nsion of microRNAs between eutopic and ectopic endometrium in\novarian endometriosis. J Biomed Biotechnol 2010;2010:369549\n7 Hu WP, Tay SK, Zhao Y. Endometriosis-specific genes identified by\nreal-time reverse transcription-polymerase chain reaction\nexpression pro filing of endometriosis versus autologous uterine\nendometrium. J Clin Endocrinol Metab 2006;91(01):228 – 238\nDoi: 10.1210/jc.2004-1594\n8 Wu Y, Kajdacsy-Balla A, Strawn E, et al. Transcriptional character-\nizations of differences between eutopic and ectopic endometrium.\nEndocrinology 2006;147(01):232– 246 Doi: 10.1210/en.2005-0426\n9 Eyster KM, Klinkova O, Kennedy V, Hansen KA. Whole genome\ndeoxyribonucleic acid microarray analysis of gene expression in\nectopic versus eutopic endometrium. Fertil Steril 2007;88(06):\n1505– 1533 Doi: 10.1016/j.fertnstert.2007.01.056\n10 Hull ML, Escareno CR, Godsland JM, et al. Endometrial-peritoneal\ninteractions during endometriotic lesion establishment. Am J\nPathol 2008;173(03):700 – 715 Doi: 10.2353/ajpath.2008.071128\n11 Revised American Society for Reproductive Medicine classi fica-\ntion of endometriosis: 1996. Fertil Steril 1997;67(05):817 – 821\nDoi: 10.1016/S0015-0282(97)81391-X\n12 Livak KJ, Schmittgen TD. Analysis of relative gene expression data\nusing real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.\nMethods 2001;25(04):402– 408 Doi: 10.1006/meth.2001.1262\n13 Goodsaid FM, Smith RJ, Rosenblum IY. Quantitative PCR decon-\nstruction of discrepancies between results reported by different\nhybridization platforms. Environ Health Perspect 2004;112(04):\n456– 460\n14 Fujii EY, Nakayama M, Nakagawa A. Concentrations of receptor for\nadvanced glycation end products, VEGF and CML in plasma,\nfollicular fluid, and peritoneal fluid in women with and without\nendometriosis. Reprod Sci 2008;15(10):1066 – 1074 Doi: 10.1177/\n1933719108323445\n15 Hotta H, Ross AH, Huebner K, et al. Molecular cloning and char-\nacterization of an antigen associated with early stages of melanoma\ntumor progression. Cancer Res 1988;48(11):2955– 2962\n16 Berditchevski F, Zutter MM, Hemler ME. Characterization of novel\ncomplexes on the cell surface between integrins and proteins\nwith 4 transmembrane domains (TM4 proteins). Mol Biol Cell\n1996;7(02):193– 207 Doi: 10.1091/mbc.7.2.193\n17 Radford KJ, Thorne RF, Hersey P. CD63 associates with transmem-\nbrane 4 superfamily members, CD9 and CD81, and with beta 1\nintegrins in human melanoma. Biochem Biophys Res Commun\n1996;222(01):13– 18 Doi: 10.1006/bbrc.1996.0690\n18 Hammond C, Denzin LK, Pan M, Grif fith JM, Geuze HJ, Cresswell P.\nThe tetraspan protein CD82 is a resident of MHC class II compart-\nments where it associates with HLA-DR, -DM, and -DO molecules.\nJ Immunol 1998;161(07):3282 – 3291\n19 Kitani S, Berenstein E, Mergenhagen S, Tempst P, Siraganian RP.\nA cell surface glycoprotein of rat basophilic leukemia cells close to\nthe high af finity IgE receptor (Fc epsilon RI). Similarity to human\nmelanoma differentiation antigen ME491. J Biol Chem 1991;266\n(03):1903– 1909\n20 Levy S, Shoham T. The tetraspanin web modulates immune-\nsignalling complexes. Nat Rev Immunol 2005;5(02):136 – 148\nDoi: 10.1038/nri1548\n21 Yoshida T, Kawano Y, Sato K, et al. A CD63 mutant inhibits T-cell\ntropic human immunode ficiency virus type 1 entry by disrupting\nCXCR4 traf ficking to the plasma membrane. Traf fic 2008;9(04):\n540– 558 Doi: 10.1111/j.1600-0854.2008.00700.x\n22 Skubitz KM, Campbell KD, Iida J, Skubitz AP. CD63 associates with\ntyrosine kinase activity and CD11/CD18, and transmits an activa-\ntion signal in neutrophils. J Immunol 1996;157(08):3617 – 3626\n23 Hirst J, Bright NA, Rous B, Robinson MS. Characterization of a\nfourth adaptor-related protein complex. Mol Biol Cell 1999;10\n(08):2787– 2802 Doi: 10.1091/mbc.10.8.2787\n24 Lekishvili T, Fromm E, Mujoomdar M, Berditchevski F. The tumour-\nassociated antigen L6 (L6-Ag) is recruited to the tetraspanin-\nenriched microdomains: implication for tumour cell motility.\nJ Cell Sci 2008;121(Pt 5):685 – 694 Doi: 10.1242/jcs.020347\n25 Latysheva N, Muratov G, Rajesh S, et al. Syntenin-1 is a new\ncomponent of tetraspanin-enriched microdomains: mechanisms\nand consequences of the interaction of syntenin-1 with CD63. Mol\nCell Biol 2006;26(20):7707 – 7718 Doi: 10.1128/MCB.00849-06\n26 Jung KK, Liu XW, Chirco R, Fridman R, Kim HR. Identi fication of\nCD63 as a tissue inhibitor of metalloproteinase-1 interacting cell\nsurface protein. EMBO J 2006;25(17):3934 – 3942 Doi: 10.1038/sj.\nemboj.7601281\n27 Schröder HM, Hoffmann SC, Hecker M, Korff T, Ludwig T. The\ntetraspanin network modulates MT1-MMP cell surface traf fick-\ning. Int J Biochem Cell Biol 2013;45(06):1133 – 1144 Doi: 10.1016/\nj.biocel.2013.02.020\n28 Toricelli M, Melo FH, Peres GB, Silva DC, Jasiulionis MG. Timp1\ninteracts with beta-1 integrin and CD63 along melanoma genesis\nand confers anoikis resistance by activating PI3-K signaling path-\nway independently of Akt phosphorylation. Mol Cancer 2013;\n12:22 Doi: 10.1186/1476-4598-12-22\n29 Jung YS, Liu XW, Chirco R, Warner RB, Fridman R, Kim HR. TIMP-1\ninduces an EMT-like phenotypic conversion in MDCK cells inde-\npendent of its MMP-inhibitory domain. PLoS One 2012;7(06):\ne38773 Doi: 10.1371/journal.pone.0038773\n30 Ron D, Chen CH, Caldwell J, Jamieson L, Orr E, Mochly-Rosen D.\nCloning of an intracellular receptor for protein kinase C: a\nhomolog of the beta subunit of G proteins. Proc Natl Acad Sci U\nS A 1994;91(03):839 – 843 Doi: 10.1073/pnas.91.3.839\n31 Berns H, Humar R, Hengerer B, Kiefer FN, Battegay EJ. RACK1 is up-\nregulated in angiogenesis and human carcinomas. FASEB J 2000;\n14(15):2549– 2558 Doi: 10.1096/fj.99-1038com\n32 Chang BY, Harte RA, Cartwright CA. RACK1: a novel substrate for the\nSrc protein-tyrosine kinase. Oncogene 2002;21(50):7619 – 7629\nDoi: 10.1038/sj.onc.1206002\n33 Cox EA, Bennin D, Doan AT, O ’ Toole T, Huttenlocher A. RACK1\nregulates integrin-mediated adhesion, protrusion, and chemo-\ntactic cell migration via its Src -binding site. Mol Biol Cell 2003;14\n(02):658– 669 Doi: 10.1091/mbc.e02-03-0142\n34 Mourtada-Maarabouni M, Kirkham L, Farzaneh F, Williams GT.\nFunctional expression cloning reveals a central role for the\nreceptor for activated protein kinase C 1 (RACK1) in T cell\napoptosis. J Leukoc Biol 2005;78(02):503 – 514 Doi: 10.1189/\njlb.0205070\n35 Viviani B, Corsini E, Binaglia M, Lucchi L, Galli CL, Marinovich M.\nThe anti-in flammatory activity of estrogen in glial cells is regu-\nlated by the PKC -anchoring protein RACK-1. J Neurochem 2002;83\n(05):1180– 1187\n36 Doan AT, Huttenlocher A. RACK1 regulates Src activity and\nmodulates paxillin dynamics during cell migration. Exp Cell Res\n2007;313(12):2667– 2679 Doi: 10.1016/j.yexcr.2007.05.013\n37 Chang BY, Conroy KB, Machleder EM, Cartwright CA. RACK1, a\nreceptor for activated C kinase and a homolog of the beta subunit\nof G proteins, inhibits activity of src tyrosine kinases and growth\nof NIH 3T3 cells. Mol Cell Biol 1998;18(06):3245 – 3256 Doi:\n10.1128/MCB.18.6.3245\n38 Mamidipudi V, Chang BY, Harte RA, Lee KC, Cartwright CA. RACK1\ninhibits the serum- and anchorage-independent growth of v-Src\nRev Bras Ginecol Obstet Vol. 40 No. 10/2018\nThe Apoptotic, Angiogenic and Cell Proliferation Genes cd63, s100a6 ,a n d gnb2l1 Gomes et al.612\n\n\ntransformed cells. FEBS Lett 2004;567(2-3):321 – 326 Doi:\n10.1016/j.febslet.2004.03.125\n39 Kiely PA, O ’ Gorman D, Luong K, Ron D, O ’ Connor R. Insulin-like\ngrowth factor I controls a mutually exclusive association of RACK1\nwith protein phosphatase 2A and beta1 integrin to promote cell\nmigration. Mol Cell Biol 2006;26(11):4041 – 4051 Doi: 10.1128/\nMCB.01868-05\n40 Zhang W, Cheng GZ, Gong J, et al. RACK1 and CIS mediate the\ndegradation of BimEL in cancer cells. J Biol Chem 2008;283(24):\n16416– 16426 Doi: 10.1074/jbc.M802360200\n41 Saito A, Fujii G, Sato Y, et al. Detection of genes expressed in\nprimary colon cancers by in situ hybridisation: overexpression of\nRACK 1. Mol Pathol 2002;55(01):34 – 39\n42 Egidy G, Julé S, Bossé P, et al. Transcription analysis in the MeLiM\nswine model identi fies RACK1 as a potential marker of malig-\nnancy for human melanocytic proliferation. Mol Cancer 2008;\n7:34 Doi: 10.1186/1476-4598-7-34\n43 Shipitsin M, Campbell LL, Argani P, et al. Molecular de finition of\nbreast tumor heterogeneity. Cancer Cell 2007;11(03):259 – 273\nDoi: 10.1016/j.ccr.2007.01.013\n44 Wang Z, Zhang B, Jiang L, et al. RACK1, an excellent predictor for\npoor clinical outcome in oral squamous carcinoma, similar to\nKi67. Eur J Cancer 2009;45(03):490 – 496 Doi: 10.1016/j.\nejca.2008.11.012\n45 Sastry M, Ketchem RR, Crescenzi O, et al. The three-dimensional\nstructure of Ca(2 þ)-bound calcyclin: implications for Ca(2 þ)-\nsignal transduction by S100 proteins. Structure 1998;6(02):\n223– 231 Doi: 10.1016/S0969-2126(98)00023-9\n46 Hwang R, Lee EJ, Kim MH, et al. Calcyclin, a Ca2 þ ion-binding\nprotein, contributes to the anabolic effects of simvastatin on bone.\nJ Biol Chem 2004;279(20):21239– 21247 10.1074/jbc.M312771200\n47 Kuźnicki J, Filipek A. Puri fication and properties of a novel Ca2 þ-\nbinding protein (10.5 kDa) from Ehrlich-ascites-tumour cells.\nBiochem J 1987;247(03):663 – 667 Doi: 10.1042/bj2470663\n48 Filipek A, Zasada A, Wojda U, Makuch R, Dabrowska R. Char-\nacterization of chicken gizzard calcyclin and examination of its\ninteraction with caldesmon. Comp Biochem Physiol B Biochem\nMol Biol 1996;113(04):745 – 752 Doi: 10.1016/0305-0491(95)\n02095-0\n49 Golitsina NL, Kordowska J, Wang CL, Lehrer SS. Ca2 þ-dependent\nbinding of calcyclin to muscle tropomyosin. Biochem Bio-\nphys Res Commun 1996;220(02):360 – 365 Doi: 10.1006/bbrc.\n1996.0410\n50 Leśniak W, Słomnicki ŁP , Filipek A. S100A6 - new facts and features.\nBiochem Biophys Res Commun 2009;390(04):1087 – 1092 Doi:\n10.1016/j.bbrc.2009.10.150\n51 Hirschhorn RR, Aller P, Yuan ZA, Gibson CW, Baserga R. Cell-cycle-\nspecific cDNAs from mammalian cells temperature sensitive for\ngrowth. Proc Natl Acad Sci U S A 1984;81(19):6004 – 6008\n52 Liu Z, Zhang X, Chen M, Cao Q, Huang D. Effect of S100A6 over-\nexpression on β-catenin in endometriosis. J Obstet Gynaecol Res\n2015;41(09):1457– 1462 Doi: 10.1111/jog.12729\n53 Matsuzaki S, Darcha C, Maleysson E, Canis M, Mage G. Impaired\ndown-regulation of E-cadherin and beta-catenin protein expres-\nsion in endometrial epithelial cells in the mid-secretory endome-\ntrium of infertile patients with endometriosis. J Clin Endocrinol\nMetab 2010;95(07):3437 – 3445 Doi: 10.1210/jc.2009-2713\n54 Zhang X, Liu Z, Chen M, Cao Q, Huang D. Effects of S100A6 gene\nsilencing on the biological features of eutopic endometrial stro-\nmal cells and β‑catenin expression. Mol Med Rep 2017;15(03):\n1279– 1285 Doi: 10.3892/mmr.2017.6105\n55 Słomnicki ŁP, Nawrot B, Leśniak W. S100A6 binds p53 and affects\nits activity. Int J Biochem Cell Biol 2009;41(04):784 – 790 Doi:\n10.1016/j.biocel.2008.08.007\n56 Tsoporis JN, Izhar S, Parker TG. Expression of S100A6 in cardiac\nmyocytes limits apoptosis induced by tumor necrosis factor-\nalpha. J Biol Chem 2008;283(44):30174 – 30183 Doi: 10.1074/\njbc.M805318200\n57 Leong S, Christopherson RI, Baxter RC. Profiling of apoptotic changes\nin human breast cancer cells using SELDI-TOF mass spectrometry. Cell\nPhysiol Biochem 2007;20(05):579– 590 Doi: 10.1159/000107541\n58 Nedjadi T, Kitteringham N, Campbell F, et al. S100A6 binds to\nannexin 2 in pancreatic cancer cells and promotes pancreatic\ncancer cell motility. Br J Cancer 2009;101(07):1145 – 1154 Doi:\n10.1038/sj.bjc.6605289\n59 Luo X, Sharff KA, Chen J, He TC, Luu HH. S100A6 expression and\nfunction in human osteosarcoma. Clin Orthop Relat Res 2008;466\n(09):2060– 2070 Doi: 10.1007/s11999-008-0361-x\n60 Ohuchida K, Mizumoto K, Ishikawa N, et al. The role of S100A6 in\npancreatic cancer development and its clinical implication as a\ndiagnostic marker and therapeutic target. Clin Cancer Res 2005;\n11(21):7785– 7793 Doi: 10.1158/1078-0432.CCR-05-0714\n61 Słomnicki LP, Le śniak W. S100A6 (calcyclin) de ficiency induces\nsenescence-like changes in cell cycle, morphology and functional\ncharacteristics of mouse NIH 3T3 fibroblasts. J Cell Biochem 2010;\n109(03):576– 584 Doi: 10.1002/jcb.22434\n62 Okada H, Sanezumi M, Nakajima T, Okada S, Yasuda K, Kanzaki H.\nRapid down-regulation of CD63 transcription by progesterone in\nhuman endometrial stromal cells. Mol Hum Reprod 1999;5(06):\n554– 558 Doi: 10.1093/molehr/5.6.554\n63 Brar AK, Handwerger S, Kessler CA, Aronow BJ. Gene induction\nand categorical reprogramming during in vitro human endome-\ntrial fibroblast decidualization. Physiol Genomics 2001;7(02):\n135– 148 Doi: 10.1152/physiolgenomics.00061.2001\n64 Tong XM, Lin XN, Song T, Liu L, Zhang SY. Calcium-binding protein\nS100P is highly expressed during the implantation window in\nhuman endometrium. Fertil Steril 2010;94(04):1510 – 1518 Doi:\n10.1016/j.fertnstert.2009.07.1667\nRev Bras Ginecol Obstet Vol. 40 No. 10/2018\nThe Apoptotic, Angiogenic and Cell Proliferation Genes cd63, s100a6 ,a n d gnb2l1 Gomes et al. 613","source_license":"CC0","license_restricted":false}