{"paper_id":"0fcf6c04-876f-45c4-8dc7-749087f5a454","body_text":"Endometriosis is a common, benign gynecologic \ndisorder recognized by the presence of the endometrial \ntissue out of the uterus, especially on pelvic organs and \nperitoneum. The most clinical presentation is pelvic pain \nworsen during menstruation, painful intercourse and \ninfertility. Endometriosis affects approximately 10% of \nwomen in reproductive age and it may occur in about \n50% of those with pelvic pain, infertility or both ( 1 ).\nSeveral theories have thus far been proposed including \nretrograde menstruation, coelomic metaplasia, steroid \nhormones, oxidative stress, impaired immune function, \ndecreased apoptosis, genetics, epigenetics, and stem \ncells, while evidences show each of these factors has \npartially been involved in endometriosis pathogenesis ( 2 ).\nDuring each menses, almost all of the functional layer\nand small amount of the basalis layer containing a lot of\nstem cells shed in the uterus ( 3 ). They can migrate out of\nthe uterus through retrograde menstruation, seed there and \nestablish endometriotic lesions. However, the presence \nof endometriosis in 10% of women despite the presence \nof retrograde menstruation in over 90% of them seems \nintriguing. Several evidences show that the stem cells\ngenerating endometriotic lesions are characteristically\ndifferent from the normal stem cells. They have a higher\nability to proliferate and a lower capacity for differentiation\nand decidualization ( 4 ). It appears to us that impaired \nproliferation/differentiation and decidualization balance \ncan changes stem cell character and function, while this \nmakes them susceptible to develop endometriosis.\nSeveral studies investigated genetic contribution in \nendometriosis, most of which failed to determine any \nsignificant correlation. Some studies demonstrated \nthat epigenetic deregulation is, in fact, the underlying \npathogenic mechanism of endometriosis ( 5 ) and it \nalters gene expression in response to hormonal and \nenvironmental factors (i.e., through dynamic changes of\nthe environment).\nEpigenetic changes play an important role in the \npathogenesis of various diseases, including cancers, \nand they are used as biomarkers for early diagnosis ( 6 ). \nEpigenetics is longtime proved concept, involved in \nstem cell regulation ( 7 ). microRNAs (miRs) are short \nnon-coding RNA molecules with critical roles in posttranscriptional \nregulation of different genes ( 8 ) and, as \nepigenetic regulators, they are key molecules involved in \nthe determination of stem cell fate by regulation of the self-\nrenewal and differentiation-related pathways ( 9 ). Their \naberrant expression can change stem cell functions and \ncause the differences between endometriotic and normal \nstem cells ( 10 ). Thus far, deregulation of microRNAs \nhas been confirmed to contribute to endometriosis and \ninfertility ( 11 ).\nIn this study, we chose three microRNAs ( miR-200b, \nmiR-145  and  let-7b ) dysregulated during endometriosis \n( 12 ) and their expressions were evaluated in endometriotic \nmesenchymal stem cells (MSCs).\nAberrant expression of  miR-200b  has been reported \nin many cancers ( 13 ). Up-regulation of this microRNA \npromotes cell proliferation in cervical cancer ( 14 ). \nTransfection of endometriotic stem cells with  miR-200b  \nincreases cell proliferation and side population phenotype \nthrough enhancing expression of  KLF4, SOX2, OCT4  \nand  c-MYC , in addition to transforming mature cells \ninto pluripotent cells ( 15 ).  miR-200c  overexpression \nin human embryonic stem cells (hESCs) up-regulates \n NANOG  expression and decreases apoptosis, resulting in \nmaintenance of their self-renewal ability and proliferation \n( 16 ).  miR-200  family helps transition of human fibroblasts \nto pluripotent stem cells by  ZEB2  suppression and \nmesenchymal-epithelial transition (MET) induction in \ncooperation with  OCT4  and  SOX2  ( 17 ).\nOverexpression of  miR-145  inhibits cell proliferation \nand migration by suppressing the  TGF-ß1  \nexpression \nin breast cancer cells ( 18 ). This microRNA induces \ndifferentiation of cervical cancer stem cells (CSCs) by \nsuppressing the stem cell transcription factors involved \nin maintaining CSCs self-renewal ( 19 ). miR-145 acts \nas a tumor suppressor molecule in a lot of cancers ( 20 ). \nmiR-145 inhibits endometriotic cell proliferation, and \nself-renewal via targeting  OCT4, KLF4,  and  SOX2  and \ninduces hESC and CSCs differentiation ( 21 - 23 ). Its \nexpression is down-regulated in hESCs and increased \nwithin differentiation.\nlet-7  is strongly accepted as a tumor suppressor \nmicroRNA and expression of its family members are \ndown-regulated in several types of cancer ( 24 ).  let-7b  \nsuppresses the expression of  OCT4  as well as SOX2 and \nit reprogrammes CSCs into the differentiated cells via a \n let-7/LIN28  feedback loop ( 25 ).  let-7b  overexpression \ninhibits proliferation and induces differentiation in adult \nand CSCs ( 26 ).\nIt seems that  miR-200b, miR-145,  and  let7b  could\nbe involved in the modulation of self-renewal and \ndifferentiation of stem cells, so their role in stem cell\ndysfunction could be postulated as a plausible theory.\nConsidering this hypothesis, we compared the \nexpression of these microRNAs ( miR-200b, miR-145,  \nand  let-7b ) in MSCs isolated from three women who \nhad pelvic endometriosis and three women without \nendometriosis. This comparison shows the aberrant \nexpression of these microRNAs in endometriotic MSCs \nand supports the presence of proliferation/differentiation \nimbalance in endometriosis initiating MSCs.\n\nThis study was approved by the Ethics Committee of \nMedical Faculty of Tarbiat Modares University (no. \n1395.409), Tehran, Iran. Written informed consent \nwas taken from each patient after a standard genetic \ncounselling.\nHuman endometrial tissue samples were obtained from \nthree premenopausal women (30-45 years old) undergoing \nhysterectomy for non-endometrial benign pathological \ncondition and another three patients with endometriosis \nundergoing laparoscopy for endometriosis in the Rasoul \nAkram Hospital of Iran Medical University (Tehran, \nIran). Eutopic endometrial tissues were obtained from the \npatients. The patients had not received hormone treatments \nfor at least three months before sample collection. \nDiagnosis of endometriotic and non-endometriotic \ncollected tissues was validated by histopathological test \nby two experienced histopathologists.\nTissues were separated and washed in phosphate \nbuffered saline (PBS) then minced into 1-2 mm3 pieces in \na medium containing Dulbecco modified Eagle medium/\nHam’s F-12 (DMEM/F-12, Invitrogen, UK) and 1% \npenicillin-streptomycin antibiotics solution (Invitrogen, \nUSA). Briefly, cell suspension of endometrial cells was \nobtained using enzymatic digestion using collagenase \ntype 3 (300 µg/ml, Sigma, Germany) and mechanical \nprocedure at 37°C for 90 minutes, then centrifuged for \n5 minutes at 3000 rpm. Cell suspensions were filtered \nthrough 150, 100, 40 mm mesh to remove undigested \ntissues and epithelial components. Endometrial stromal \ncells were next cultured in DMEM/F-12 containing 1% \npenicillin-streptomycin solution and 10% fetal bovine \nserum (FBS, Gibco, USA) at 37°C in 95% air and 5% \nCO 2  conditions. Endometrial stromal cells in passages \n3-4 were used for characterization by flow cytometry \nanalyses.\nIsolated stromal cells were trypsinized and centrifuged.\nThe cell pellet was resuspended in PBS supplemented with \n5% FBS and incubated with monoclonal antibodies for 30 \nminutes at 4°C in the dark. Human CD45 (BD Bioscience, \nUSA) and CD34 (IMMUNOSTEP, Spain) antibodies \nwere served as negative controls, while anti-human \nCD90 (BD Bioscience, USA), CD105 (IMMUNOSTEP, \nSpain), CD73 (BD Bioscience, USA) and CD146 (BD \nBioscience, USA) were used as specific antibodies. Cells \nwere evaluated with a FACS Calibur apparatus (Becton \nDickinson, USA). Finally, the analysis was done using \nFlowJo 7.6 software.\nFor evaluating the endometrial MSCs differentiation \npotential, endometrial stromal cells (CD146+, CD90+, \nCD105+, CD73+ and CD34-, CD45-) were seeded in \n24-well plates and cultured in osteogenic and adipogenic \ndifferentiation media for 4 weeks, separately. Control cells \nwere also cultured in low serum medium (DMEM/F12 \nwith 1% FBS and 1% penicillin-streptomycin antibiotic \nsolution) for the same incubation time. Control and \ndifferentiation media were changed every 2-3 days. Three \nweeks later, osteogenic and adipogenic differentiations \nwere respectively checked by staining with 4% Alizarin \nRed (pH=4.1) and 1% Oil Red O (both from Sigma, \nGermany) ( 27 ).\nTotal RNA was extracted from the cells using TRIzol \nreagent (Sigma, Germany). RNA concentration and purity \nwere assessed by Nanodrop (the ratio of absorbance at \n260 and 280 nm =1.8), then we ran the extracted RNA \non denaturing agarose gel electrophoresis and the gel was \nstained with ethidium bromide for evaluating the quality \nof extracted RNA. cDNA was synthesized using specific \nstem-loop primers for microRNAs ( miR-200b, let-7b,\nmiR-145,   and  RNU44 ) in a total volume of 20 µl using the \ncDNA synthesis kit (Takara Bio, Japan).\nStem-loop RT primers were designed in accordance \nwith the protocol described by Chen et al. ( 28 ). Primer \nsequences are presented in Table 1.\nTo determine expression of the microRNAs ( miR-200b, \nmiR-145  and  let-7b ) in the cells, we used the Allele ID6 \nand Oligo7 software for designing the specific forward \nprimers and universal reverse primer. RNU44 was \nused as an internal control. Primers were synthesized \nat Pishgam Co. (Tehran, Iran). We used Syber Green \nAssay kit (Applied Biosystems, UK) according to the \nmanufacturer’s protocol. qRT-PCR reactions were done \nin 10 µl of the reaction mixture using AB StepOne Real-\nTime PCR System (Applied Biosystems, UK). All qRT-\nPCR experiments were repeated three times. Data were \nanalyzed using Pfaffl method and normalized by  RNU44  \nexpression in each sample.\nWe used student’s t test by GraphPad Prism 6 software \nfor statistical analysis and comparison of microRNA \nexpressions between samples. Results were considered \nsignificant at P<0.05.\n\nHuman MSCs were isolated from the endometrium and \nthey were cultured. Flow cytometry analysis confirmed \nthe expression of MSC markers CD73 (98.5%), CD90 \n(99.1%), CD105 (96.3%) and CD146 (84.8%). Expression \nof hematopoietic markers, including CD34 (0.474%) and \nCD45 (1.99%), were negative ( Fig .1A-F ). To evaluate \ndifferentiation potential of the isolated endometrial MSCs, \nwe induced adipogenic and osteogenic differentiation with \nspecific differentiation media, as specified. Confirmation \nof differentiation was done through staining of calcium \ndeposits by alizarin red and lipid vacuoles through oil red \nstaining ( Fig .1G, H ).\nSequence of oligonucleotide primers used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) measurements\nIsolation and characterization of endometrial mesenchymal stem cells (MSCs). Flow cytometry analyses showed that endometrial MSCs positively \nexpressed  A.  CD73 (98.5%),  B.  CD90 (99.1%),  C.  CD105 (96.3%),  D.  CD146 (84.9%) but negatively expressed,  E.  CD34 (0.474%),  F.  CD45 (1.99%), G. \nOsteogenic, and  H.  adipogenic differentiation of the isolated endometrial MSCs.\nTo explore microRNAs profiling in endometrial MSCs \nof the endometriotic and non-endometriotic control \ngroups the expression levels of  miR-200b, miR-145  and \n let-7b  were evaluated by qRT-PCR. The efficiency of \nqRT-PCR reactions for  miR-200b, miR-145  and  let-7b  \nwere measured using LinReg software algorithm ( 29 ). \nEach experiment was repeated three times to eliminate \nany subjective variation. All reactions were assessed for \ndistinct melting curves, while they showed no nonspecific \nor primer-dimer peaks.\nRelative expressions of miR-200b in the endometriotic \nMSCs showed up-regulation of this microRNA (4.199 \n± 0.6617, P<0.0001) in comparison with the nonendometriotic \ncontrol group ( Fig .2A ).\nExpression of miR-145 in the endometriotic MSCs was \ndecreased to 0.5467 ± 0.06137 fold (P<0.0001) in comparison \nwith the non-endometriotic control group ( Fig .2B ).\nExpression of  let-7b  in the endometriotic MSCs was \n0.3024 ± 0.04454 fold (P<0.0001) less than the nonendometriotic \ncontrol group ( Fig .2C ).\nmicroRNA expression analyses. Relative expressions of miR200b, \nmiR-145 and let-7b in endometrial mesenchymal stem cells \n(MSCs) of endometriotic patients and non-endometriotic control \ngroup, evaluated by quantitative reverse transcription polymerase \nchain reaction (qRT-PCR). A. miR-200b expression in endometritic \nMSCs was 4.199 ± 0.6617 fold (P<0.0001) higher than nonendometriotic \nMSCs, B. miR-145 expression in endometritic MSCs \nwas 0.5467 ± 0.06137 fold (P<0.0001) less than non-endometriotic \nMSCs, and C. Expression of let-7b in endometriotic MSCs was 0.3024 \n± 0.04454 fold (P<0.0001) less than non-endometriotic MSCs control \ngroup. ****; P<0.0001 in comparison to non-endometriotic MSCs.\n\nWe believe that proliferation/differentiation imbalance \nplays a pivotal role in the pathogenesis of endometriosis. \nWe evaluated  miR-200b, miR-145  and  let-7b  expression \nas modulators of stem cell proliferation and differentiation \n( 30 ), in endometriotic and non-endometriotic MSCs. \nPrevious studies have shown that these microRNAs were \nderegulated in endometriosis while no study evaluated \ntheir expression in endometriotic MSCs ( 31 ,  32 ).\nSeveral theories are proposed as the pathogenesis basis\nof endometriosis. stem cell theory is one main research\nfield in endometriosis. A lot of studies described the role \nof stem cells in endometriosis development ( 3 ). A balance\nin proliferation/differentiation equilibrium is required for \nthe correct function of stem cells and it seems to us that in\nseveral diseases including endometriosis, this balance fails, \nresulting in altered function of stem cells, and changing \ntheir fate. Many studies confirm that endometriotic \nMSCs are different from non-endometriotic types. They \nhave a higher ability to migrate, attach and proliferate \n( 33 ,  34 ), while a lower capacity for differentiation and \ndecidualization is proposed for them, due to the impaired \ndecidualization related pathways ( 4 ). We believe that\nproliferation/differentiation imbalance in endometriotic\nMSCs is the main underlying cause for endometriosis \ndevelopment and its correlated infertility.\nPrevious studies have shown that microRNAs are \ninvolved in regulation of signaling pathways that control \ndifferentiation and proliferation of stem cells during \nnormal development and disease pathogenesis ( 30 ).\nmiR-200b, miR-145  and  let-7b  are deregulated in several \ndiseases like cancers confirming the aforementioned \nimbalance. These microRNAs have specific expression \nprofile in endometrial stromal cells during decidualization \n( 35 ). Deregulation of these microRNAs has been shown \nin the ectopic and eutopic endometrium of women with \nendometriosis, but our study is the first to confirm their \nexpressions and roles in endometriotic MSCs. We find \nthat in endometriotic MSCs  miR-200b  is up-regulated \nsignificantly as compared to normal control group. Previous \nstudies have shown that  miR-200b  is up-regulated in eutopic \nendometrium of endometriotic women and involved in \nendometriosis-associated infertility ( 31 ). Overexpression of \n miR-200b  increases cell proliferation and MET. It induces \ngeneration of pluripotent stem cells in cooperation with \ntranscription factor  SOX2  and  OCT4  ( 17 ).\nTransfection of endometriotic stem cells with  miR-200b  \nresults in increase side population phenotype through \nactivating  KLF4  and  NANOG  expressions as well as \nMET, while reducing decidualization. It also enhances \nmetastatic colonization of successfully migrated cells by \ninhibiting secretion of metastasis inhibitors ( 15 ).  miR200  \nfamily members are down-regulated during  in vitro  \ndecidualization ( 35 ).\nIncreased expression of  miR-200b  in endometriotic \nMSCs, in our study, is in accordance with the findings\nof previous studies. It might increase colonization chance \nof the migrated stem cells, enhance their proliferation \nand promote their stemness properties by positive\nregulation of stemness-related genes while decreasing\nthe differentiation potential and decidualization. These \nchanges promote development of endometriosis, disrupt\nembryo implantation and cause infertility.\nOur findings show that  miR-145  is down-regulated in \nendometriotic MSCs. Previous studies have demonstrated \nthat  miR-145  is down-regulated in the serum of \nendometriotic patients in comparison with normal control \nand potentially served as noninvasive biomarkers for \nendometriosis. Transfection of endometrial stromal cells \nwith  miR-145  inhibits cell proliferation and invasiveness. \nIt also suppresses the stemness by down-regulation of \nstemness-related genes ( 36 ). This microRNA induces \ndifferentiation of stem cells through SOX2-LIN28/let7 \nsignaling pathway by decreasing SOX2 and LINE-28 \nprotein levels ( 37 ). Overexpression of this microRNA \nin CSCs reduces the expression of stemness-related \nmarkers, while it increases cancer cells differentiation \n( 38 ). In the present study, decreased level of  miR-145  \nin endometriotic MSCs confirms findings obtained from \nprevious studies. This is consistent with the underlying \nproposed pathogenesis mechanism to increase stem cell \nproliferation, decrease their differentiation and facilitate \nendometriosis risk.\nOur results show a down-regulation of  let-7b  in the \nendometrial MSCs of women with endometriosis. \nPrevious studies have also shown that expression of  let-7  \nwas decreased in the serum of endometriotic patients in \ncomparison with normal control ( 32 ).\nlet-7  is involved in a regulatory feedback loop \nwith LIN28, which has a critical role in pluripotency \nmaintenance in collaboration with  NANOG, SOX2  and \n OCT4  genes. Overexpression of this microRNA in stem \ncells promotes differentiation, while inhibition of  let-7  \nresults in the proliferation of stem cells and decreases \ndifferentiation. Briefly,  let-7b  family members fine-\ntune the pathways related to self-renewal/differentiation \nbalances ( 39 ).\nlet-7  suppresses the expression of  OCT4  and  SOX2 . \nIt reprogrammes CSCs to differentiate via  let-7/LIN28  \nfeedback loop and its overexpression regulates the \nstemness by increasing differentiation and decreasing \nself-renewal in both of the normal and cancer stem \ncells ( 26 ) Reduced level of  let-7  is required for self-\nrenewal and maintenance of the undifferentiated state of \nembryonic and adult stem cells and its overexpression has \nopposing effects, reducing their proliferation and leading \nto their differentiation ( 39 ). Overexpression of  let-7b  \nin neural stem cells inhibits proliferation and promotes \ndifferentiation ( 40 ).\nIn this study,  let-7b  down-regulation in endometriotic \nMSCs consolidates the results of previous studies.  let- 7b  \nis proposed as one of the main players of proliferation/ \ndifferentiation imbalance in endometriotic MSCs. In \nother words, any deregulation of  let-7b  expression alters\nproliferation/differentiation balance in endometriotic \nMSCs.  let-7b  deregulation increases the probability \nof endometriotic lesion formations via enhancing the \nstem cell proliferation, migration, self-renewal and\nmaintenance of their undifferentiated state. These changes\nreduce decidualization and increase infertility in patients\nwith endometriosis.\nAlthough the exact underlying pathologic mechanism \nof endometriosis is yet unclear, current findings \ndiscover the strong role of stem cells in endometriosis \nand confirm their different characteristics and function. \nOur results consolidate the theory of imbalance \nbetween differentiation and proliferation capacity, \nespecially in stem cells of endometriotic patients. \nThis study for the first time evaluates the expression \nof  miR-200b, miR-145  and  let-7b  in endometrial MSCs \nof women with endometriosis in comparison with \nnormal control, representing that aberrant expression \nof these microRNAs is present in this pathological \ncondition. These microRNAs contribute to modulating \nproliferation and/or differentiation of stem cells. This \nis the first study to evaluate the expression of these \nmicroRNAs in endometriotic stem cells. Our findings \nare in support of a unified differentiation/proliferation \nimbalance theory.\n\nEndometriosis is a complex and yet unknown \ngynecological disease in women. Deregulation of \nmicroRNAs related to differentiation and proliferation \nin endometriotic MSCs compared to the normal types \nconfirms the implication of epigenetics and this is \nin line with many other authors, while supporting \nthe underlying mechanism of endometriosis, as \nemphasized in this study. We think that impaired \nbalance between differentiation and proliferation in \nMSCs, which is supported by our study, is essential \nfor endometriosis development.","source_license":"CC0","license_restricted":false}