{"paper_id":"6f5f0d1a-eb7a-4368-ab58-9af65b265d3c","body_text":"Liu et al. Cell Biosci            (2019) 9:84  \nhttps://doi.org/10.1186/s13578-019-0346-3\nRESEARCH\nLncRNA H19 over-expression inhibited Th17 \ncell differentiation to relieve endometriosis \nthrough miR-342-3p/IER3 pathway\nZheying Liu†, Liya Liu†, Yun Zhong, Mingbo Cai, Junbi Gao, Chaoyue Tan, Xiaoxiao Han, Ruixia Guo \nand Liping Han*\nAbstract \nObjective: To investigate the mechanism of LncRNA H19 in Th17 cell differentiation and endometrial stromal cells \n(ESCs) proliferation in endometriosis (EMS).\nMethods: LncRNA H19, miR-342-3p and IER3 expressions were detected by qRT-PCR and western blot. The percent-\nage of Th17 cells/CD4+ T cells was detected by flow cytometry. IL-17 level was measured by ELISA. The interaction of \nmiR-342-3p and IER3 was confirmed by Luciferase reporter assay.\nResults: LncRNA H19 and IER3 expressions were down-regulated in mononuclear cells from peritoneal fluid (PFMCs) \nof patients with EMS or under Th17 differentiation conditions, whereas miR-342-3p expression was up-regulated and \nthe percentage of Th17 cells was increased in PFMCs of patients with EMS or under Th17 differentiation conditions. \nOver-expression of LncRNA H19 decreased IL-17 level and the percentage of Th17 cells/CD4+ T cells. Besides, we \nconfirmed that miR-342-3p could target to IER3 and negatively regulate IER3 expression. LncRNA H19 over-expression \nsuppressed Th17 differentiation and ESC proliferation through regulating miR-342-3p/IER3. In vivo experiments \nshowed LncRNA H19 over-expression suppressed the growth of Th17 cell differentiation-induced endometriosis-like \nlesions.\nConclusion: LncRNA H19 was down-regulated in PFMC of patients with EMS or under Th17 polarizing conditions, \nand LncRNA H19 over-expression suppressed Th17 cell differentiation and ESCs proliferation through miR-342-3p/IER3 \npathway.\nKeywords: LncRNA H19, miR-342-3p, IER3, Th17 cell differentiation, Endometriosis\n© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License \n(http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, \nprovided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, \nand indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/\npublicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.\nIntroduction\nEndometriosis (EMS) is a common gynecological disor -\nder characterized by the presence of endometrial tissue \noutside the uterine cavity, which affects approximately \n10% of reproductive-aged women and causes pain and \ninfertility [1]. Although this disease has been investi\n-\ngated for decades, the pathogenesis of EMS is still not \nfully understood. Sikora et  al. have found that retro\n-\ngrade menstruation forms endometriosis on the basis of \nendometrial fragments reaching the pelvis via transtubal \nretrograde flow and causes chronic inflammation [2]. T \nhelper cell 17 (Th17) is a subset of T cells differentiated \nfrom CD4+ T cells that can secrete interleukin 17 (IL-\n17) and play important roles in autoimmune disease and \ndefensive response [3]. IL-17 family members can induce \ninflammation, activate dendritic cells or macrophages, \nand promote tissue inflammation [4]. Researchers stud\n-\nied the phenotype of Th17 cells and found an elevation \nof inflammatory cytokines IFN-γ, IL-17A, TNF-α, IL-1β \nin the peritoneal fluid (PF) of patients with endome\n-\ntriosis, and the percentage of Th17 cells was remarkably \nincreased in S (I-II) and S (III-IV) stages of EMS [5]. \nOpen Access\nCell & Bioscience\n*Correspondence:  hanliping0825@163.com\n†Zheying Liu and Liya Liu contributed equally to this work\nDepartment of Gynecology, The First Affiliated Hospital of Zhengzhou \nUniversity, No. 1 Jianshe Dong Road, Zhengzhou 450052, People’s \nRepublic of China\n\nPage 2 of 10Liu et al. Cell Biosci            (2019) 9:84 \nTherefore, we focus on Th17 cells and try to find the sign-\naling pathways that regulate Th17 cells in EMS.\nImmediate early response gene (IER3), also called IEX-\n1, belongs to the group of genes rapidly activated during \ninflammation that functions as an apoptosis inhibitor in \nTNF-induced apoptosis and extends duration of immune \nresponses [6, 7]. Zhi et al. proved that lack of IEX-1 pro\n-\nmoted Th17 differentiation and increased IL-17 pro -\nduction [8]. So, we assume that IER3 might involve in \nthe signaling pathways that regulate Th17 cells in EMS. \nMicroRNAs act as a post-transcriptional regulatory func\n-\ntion through binding to the 3′-untranslated region (UTR) \nof the target genes in EMS [9, 10]. It has been found that \nmiR-342-3p is highly expressed in serum of woman with \nEMS through microarray profiling [11]. Bioinformatics \nsoftware predicts IER3 is one of the target genes of miR-\n342-3p. Therefore, miR-342-3p may involve in the regula\n-\ntion of Th17 cells in EMS through targeting IER3.\nLong non-coding RNAs are RNAs longer than 200 \nnucleotides that involve in post-transcriptional and epi -\ngenetic regulation in various biological processes [12, 13]. \nLncRNA H19 is one of the most widely studied LncRNAs \nin cancers, angiogenesis, diabetes mellitus, etc. [14, 15]. \nIn 2010, H19 was firstly detected in women with EMS \nand found lower expression in endometrial tissue of \ninfertility cases than fertile cases [16]. Recently, research\n-\ners have found H19 expression is remarkably decreased \nin the eutopic endometrium of women with EMS, and \ndiscover its mechanism in reducing the proliferation of \nendometrial stromal cells (ESCs) [17]. Moreover, Wang \net  al. proved that H19 interacted with miR-342-3p and \nnegatively modulated miR-342-3p in gallbladder cancer. \nHence, we speculate LncRNA H19/miR-342-3p/IER3 is \ninvolved in the regulation of Th17 cells in EMS.\nIn this study, we showed that LncRNA H19 was down-\nregulated in mononuclear cells from peritoneal fluid \n(PFMCs) of patients with EMS, and found over-expres\n-\nsion of H19 decreased the secretion of IL-17 and reduced \nthe percentage of Th17 cells/CD4+ T cells through miR-\n342-3p/IER3, thereby contributing to suppress Th17 dif\n-\nferentiation and ESC proliferation.\nMaterials and methods\nPreparation of peritoneal fluid samples\nPeritoneal fluid samples were collected from patients \nwith regular menstrual cycles who underwent a salpingo-\noophorectomy or evisceration for the treatment of ovar\n-\nian endometriotic cysts without any hormonotherapy for \nat least 3 months prior to operation (EMS group, n = 20) \nand premenopausal patients who underwent hyster\n-\nectomies for subserousal leiomyoma without evidence \nof endometriosis (control group, n = 16) under sterile \ncondition after the laparoscopy in order to minimize \nblood contamination. The protocols were approved by \nthe Ethics Committee of The First Affiliated Hospital of \nZhengzhou University (2019-KY-197). All patients signed \ninformed consent.\nPreparation and identification of PFMCs\nPFMCs were prepared and identified according to pre -\nvious report [18]. Peritoneal fluid samples collected \nfrom EMS and control groups as described above were \ncentrifuged at 200g for 5  min, and the supernatant was \nremoved. Cell pellets were re-suspended in phosphate \nbuffered saline (PBS), and isolated by Histopaque-1077 \n(Sigma, USA) according to the manufacturer’s instruc\n-\ntions. Cells were centrifuged at 150g for 30 min, and col -\nlected at the interface. For the identification of PFMCs \n(purity > 97%), indirect immunofluorescence (IIF) was \nconducted. Anti-CD3 (Abcam, USA), anti-B19 (Abcam, \nUSA), anti-CD56 (Invitrogen, USA), and anti-CD14 \n(Abcam, USA) monoclonal antibodies were used to iden\n-\ntify T lymphocytes, B lymphocytes, natural killer lym -\nphocytes, and macrophages.\nIsolation and purification of CD4+ T cells\nPeripheral blood mononuclear cells (PBMCs) were \ncollected from healthy fertile women and isolated by \nHistopaque-1077 (Sigma, USA) according to the manu\n-\nfacturer’s instructions, washed twice with RPMI-1640 \nmedium (Gibco, USA), counted by a Neubauer hemo\n-\ncytometer, and re-suspended at 1 × 106  cells/mL. Mag -\nniSort™ Human CD4 T cell Enrichment Kit (Invitrogen, \nUSA) was used to isolate CD4+ T cells according to the \nmanufacturer’s instructions (purity > 95%). Naïve CD4+ \nT cells were isolated using MagniSort Human CD4 Naive \nT cell Enrichment Kit (eBioscience, USA). The proto\n-\ncols were approved by the Ethics Committee of The First \nAffiliated Hospital of Zhengzhou University. All patients \nsigned informed consent.\nFor CD4+ T cell transfection, lentivirus-mediated H19 \nover-expression (lenti-H19), lentivirus-mediated miR-\n342-3p mimic, lentivirus-mediated miR-342-3p inhibitor \nlentiviral vectors and scramble sequence was set as nega\n-\ntive control.\nTh17 polarization induction\nCD4+ T cells differentiation into Th17 cells were per -\nformed according to previous report [19]. CD4+ T cells \n(5 × 10\n5) were incubated for 48  h with anti-CD3 (1  μg/\nmL) (Abcam, USA), anti-CD28 antibody (1  μg/mL) \n(Abcam, USA), IL-1β (20  ng/mL)(Gibco, USA), IL-6 \n(20 ng/mL) (Gibco, USA), IL-23 (20 ng/mL) (Invitrogen, \nUSA), IFN-γ-neutralizing antibody (2 μg/mL) (Cell Sign\n-\naling Technology, USA), and IL-4-neutralizing antibody \n(2 μg/mL) (Cell Signaling Technology, USA).\n\nPage 3 of 10\nLiu et al. Cell Biosci            (2019) 9:84 \nQuantitative real‑time RCR (qRT‑PCR)\nTotal RNAs from PFMCs and CD4+  T cells were \nextracted by Trizol (Invitrogen, USA), and inversely \ntranscribed into cDNA using the High-Capacity cDNA \narchive kit (Invitrogen, USA). qRT-PCR was conducted \nto measure H19 and miR-342-3p expression using Pow\n-\nerUp™ SYBR ™ Green Master Mix (Invitrogen, USA). \nThe relative expressions of H19 and miR-342-3p were \nexpressed as a function of threshold cycle (Ct) and ana\n-\nlyzed by  2− ΔΔCt  method. Specific primers for H19 and \nmiR-342-3p were as follows: H19, F: 5′ -GCT CCA CTG \nACC TTC TAA AC-3′; miR-342-3p, F: 5′ -UCU CAC ACA \nGAA AUC GCA CCCGU-3′.\nWestern blot\nPFMCs and CD4+  T cells were lysed in Radio Immu -\nnoprecipitation Assay (RIPA) buffer (Beyotime, China). \nProtein samples (50  ng) was separated by SDS-poly\n-\nacrylamide gel electrophoresis (PAGE) and transferred \nto polyvinylidene fluoride (PVDF) membrane (Invitro\n-\ngen, USA). The membrane was incubated with primary \nantibodies against IER3 (Invitrogen, USA), β-actin \n(Abcam, USA) and horseradish peroxidase-conjugated \nsecondary antibody (Abcam, USA). Blots were detected \nby enhanced chemiluminescence, and band intensities \nwere quantified using image software Image Lab (Bio-\nRad, USA). β-actin was used as an internal control.\nFlow cytometry\nPFMCs or CD4+  T cells were collected and re-sus -\npended at 2 × 106 cells/mL. Cells were detected by BD \nFACSCanto II flow cytometry (BD, USA) and analyzed \nusing CELLQuest software. Cells positive for both CD4 \nand intercellular IL-17A were considered as Th17. Cells \nwere collected and incubated with APC-conjugated \nanti-CD4 antibody (Invitrogen, USA), anti-IL-17A anti\n-\nbody (Invitrogen, USA) and anti-IFN-γ (Invitrogen, \nUSA) for the observation of Th17 cells.\nEnzyme‑linked immuno sorbent assay (ELISA)\nThe cytokine IL-17 level from CD4+  T cell culture \nsupernatant was detected by the IL-17A Human ELISA \nKit (Invitrogen, USA).\nLuciferase reporter assay\nThe sequence of IER3 3′ UTR or the mutated sequence \nwas predicted to interact with miR-342-3p and inserted \ninto pGL3 vector (Promega, USA) which were noted \nas IER3 3′UTR wild-type (WT) or IER3 3′ UTR mutant \n(MUT) that constructed by Sangon Biotech (Shang\n-\nhai, China). The reporter plasmid (IER3 3′ UTR WT \nor IER3 3′ UTR MUT, 500  ng) and microRNAs (miR-\n342-3p mimic, miR-342-3p inhibitor, or negative \ncontrol, 1000  ng) were transfected into HEK293 cells \nfor 48 h for Luciferase reporter assay. Luciferase activ\n-\nity was measured by dual Glo ™ Luciferase Assay System \n(Promega).\nIsolation and culture of ESC\nESCs were isolated and cultured according to previ -\nous report [5]. Endometrial tissues were collected from \npatients with regular menstrual cycles but without endo\n-\nmetriosis and/or adenomyosis who underwent a hyster -\nectomy for the treatment of uterine leiomyoma under \nsterile conditions and none of the included patients had \nexperienced hormonotherapy. Tissues were digested \nwith 0.1% collagenase type IV (Sigma, USA) at 37  °C \nfor 30  min with constant agitation. Then, sterile gauzes \n(pore diameter size: 200 mesh) was used to filtrate the \ntissue pieces to remove debris. The supernatant was dis\n-\ncarded during gentle centrifugation, and the cells were \nre-suspended in DMEM/F-12 medium (Gibco, USA). \nFor removal of epithelial cells, ESCs were passed through \nsterile gauzes (pore diameter size: 400 mesh). For removal \nof leukocytes and erythrocytes, the filtrated suspension \nwas layered over Ficoll, and centrifuged at 800×g for \n20 min. The middle layer was collected and washed with \nD-Hanks solution. Then, ESCs were placed in a culture \nflask, and allowed to adhere for 20  min. The adherent \nstromal cells were cultured as monolayer in flasks with \nDMEM/F-12 (Gibco, USA) containing 10% Fetal bovine \nserum (Gibco, USA), and incubated in 5%  CO\n2 incuba -\ntor at 37 °C. The protocols were approved by the Ethics \nCommittee of The First Affiliated Hospital of Zhengzhou \nUniversity. All patients signed informed consent.\nCo‑culture of CD4+ T cells and ESC\nTo establish ESC and CD4+ T cells co-culture unit, ESCs \nwere cultured in 48-well plates at a concentration of \n2 × 10\n5 cells/well until they adhered to the plastic. Then, \nthe media were removed, and CD4+ T cells with differ -\nent treatment were applied over ESCs at the same con -\ncentration for 48 h.\nMTT assay\n3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium \nbromide (MTT) assay was used to detect ESC prolifera\n-\ntion. ESCs (2 × 104) were seeded into a 96-well plate and \nincubated overnight. 20 μL MTT (5 mg/mL; Invitrogen, \nUSA) was added to each well and cultured for 4 h. Then, \ncells were lysed using dimethylsulfoxide (150  μL/well; \nSinopharm Chemical Reagent, China). The optical den\n-\nsity was read at 570 nm.\n\nPage 4 of 10Liu et al. Cell Biosci            (2019) 9:84 \nResazurin assay\nResazurin assay was also used to detect ESC prolifera -\ntion according to previous report [20]. ESCs (2 × 104) \nwere seeded into a 96-well plate and incubated over -\nnight. 20  μL resazurin solution (0.1 mg/mL; Invitrogen, \nUSA) was added to each well and cultured at 37  °C for \n4 h. Fluorescence intensity was monitored, the excitation \nat 530 nm and emission at 590 nm was measured using a \nmicroplate reader (Thermo Scientific, USA).\nIntraperitoneal endometriosis model\nThe nude mouse endometriosis model was established \naccording to previous report [5]. The nude mice (8 week-\nold) and female C57BL/6 mice were purchased from \nLaboratory animal center of Zhengzhou University. The \nanimal experiments were approved by Ethics Committee \nof The First Affiliated Hospital of Zhengzhou University. \nNude mice were used to construct an allotransplanta\n-\ntion of intraperitoneal endometriosis model. At 0th day, \nthe uterus of female C57BL/6 mice was minced, and the \ntissue debris was intraperitoneally injected into the nude \nmice. At 5th day, CD4+ T cells from female C57BL/6 mice \nwere transfected with lenti-NC or lenti-H19, induced for \nTh17 polarization and transferred to the abdominal cav\n-\nity in endometriosis nude mice. So the nude mice were \ndivided into EMS, EMS + Th17, EMS + lenti-NC + Th17, \nand EMS + lenti-H19 + Th17 groups, with six mice in each \ngroup. At 14th day, the nude mice were sacrificed, and the \nendometriosis-like lesions and PF were collected for the \ndetection of lesion weight, and LncRNA H19, miR-342-3p \nand IER3 expressions.\nStatistical analysis\nAll data were presented as mean ± standard deviation and \nanalyzed by SPSS software (Version 15.0, USA). Each experi-\nment was repeated for three times. The differences between \ngroups were assessed by t-test or one-way analysis of variance \n(ANOVA), with p < 0.05 considered statistically significant.\nResults\nLncRNA H19 was down‑regulated in PFMC of patients \nwith EMS\nWe first examined the expressions of LncRNA H19, \nmiR-342-3p and IER3 in PFMCs in patients with EMS. \nAs shown in Fig.  1a, LncRNA H19 expression was sig\n-\nnificantly down-regulated in PFMC of EMS group than \nFig. 1 LncRNA H19 was down-regulated in mononuclear cells from peritoneal fluid (PFMC) of patients with endometriosis (EMS). PFMCs were \nisolated from patients with EMS (n = 20) and controls (n = 16). a qRT-PCR showed that LncRNA H19 was down-regulated and miR-342-3p was \nup-regulated in PFMC of EMS group than that of control group. b Western blot showed protein level of IER3 was down-regulated in PFMC of EMS \ngroup. c Flow cytometry showed the percentage of IL17 + Th17/PMSCs was higher in PFMC of EMS group than that of control group. *p < 0.05, \ncompared with control\n\nPage 5 of 10\nLiu et al. Cell Biosci            (2019) 9:84 \nthat of control group, and miR-342-3p expression was \nsignificantly up-regulated in PFMC of EMS group than \nthat of control group. Protein level of IER3 was signifi\n-\ncantly down-regulated in PFMC of EMS group than that \nof control group (Fig.  1b, Additional file  1: Figure S1B). \nWe also observed the percentage of IL17 + Th17 cells in \nPFMCs was higher in EMS group than that of control \ngroup (Fig. 1c).\nLncRNA H19 was down‑regulated in Th17 differentiation \nconditions\nNext, we isolated CD4+ T cells from healthy patients and \ninduced Th17 polarization for 48  h under Th17 polar -\nized conditions. After confirming the induction is suc -\ncessful, we tested the expressions of H19, miR-342-3p \nand IER3. We found the percentage of Th17 cells/CD4+ \nT cells was significantly increased in induced group than \nFig. 2 LncRNA H19 was down-regulated in Th17 polarizing conditions. CD4+ T cells from healthy patients were incubated under appropriate \nconditions to induce Th17 polarizing for 48 h. a The percentage of Th17 cells/CD4+ T cells was higher in induced group than that of control group. \nb LncRNA H19 was down-regulated, miR-342-3p was up-regulated and IER3 was down-regulated in induced group than that of control group. c \nELISA assay showed that IL-17 level was increased in induced group than that of control group. *p < 0.05, compared with control\nFig. 3 LncRNA H19 over-expression suppressed Th17 differentiation. CD4+ T cells were transfected with H19 over-expression lentiviral vectors \nor negative controls. a LncRNA H19 was up-regulated in CD4+ T cells transfected with H19 over-expression lentiviral vectors. b IL-17 level was \ndecreased in CD4+ T cells transfected with H19 over-expression lentiviral vectors. c TH17 cell marker RORγt expression in Lenti-NC and Lenti-H19 \ngroups was detected by western blot. The percentage of Th17 cells/CD4+ T cells was decreased in lenti-H19 group. *p < 0.05, compared with \nlenti-NC\n\nPage 6 of 10Liu et al. Cell Biosci            (2019) 9:84 \nthat of control group (Fig.  2a). The percentage of Th17 \ncells/naïve CD4+ T cells was also significantly increased \nin induced group than that of control group (Additional \nfile  1: Figure S1A). LncRNA H19 expression was sig\n-\nnificantly down-regulated, miR-342-3p expression was \nsignificantly up-regulated, and IER3 protein level was sig\n-\nnificantly down-regulated in induced group than that of \ncontrol group (Fig.  2b). IL-17 level was also significantly \nincreased in induced group than that of control group \n(Fig. 2c).\nLncRNA H19 over‑expression suppressed Th17 \ndifferentiation\nTo observe the effect of LncRNA H19 on Th17 dif -\nferentiation, CD4+ T cells were transfected with H19 \nover-expression lentiviral vectors. As shown in Fig.  3a, \nLncRNA H19 expression was significantly up-regulated \nin CD4+ T cells transfected with H19 over-expression \nlentiviral vectors than that of Lenti-NC group. IL-17 level \nwas significantly decreased in lenti-H19 group than that \nof Lenti-NC group (Fig. 3b). Then, CD4+ T cells in Lenti-\nNC group and Lenti-H19 group were under an induced \nTh17 polarizing condition. We found TH17 cell marker \nRORγt expression was down-regulated in Lenti-H19 \ngroup than Lenti-NC group (Fig.  3c), and the percentage \nof Th17 cells/CD4+ T cells was decreased in lenti-H19 \ngroup than Lenti-NC group (14.84% vs 7.86%) (Fig.  3c). \nThese data proved that LncRNA H19 over-expression \nsuppressed Th17 differentiation.\nLncRNA H19 over‑expression regulated miR‑342‑3p/IER3 \nexpression\nAccording to the prediction of bioinformatics software, \nmiR-342-3p could target to 3′UTR of IER3 (Fig.  4a). \nNext, miR-342-3p mimic or inhibitor was transfected \ninto HEK293 cells to detect the luciferase activity. miR-\n342-3p mimic significantly decreased the luciferase activ\n-\nity of IER3-WT, and there was no significant change in \nthe luciferase activity of IER3-MUT. miR-342-3p inhibi\n-\ntor significantly increased the luciferase activity of IER3-\nWT, and there was no significant change in the luciferase \nactivity of IER3-MUT. mRNA and protein level of IER3 \nwas down-regulated by miR-342-3p mimic, and up-reg\n-\nulated by miR-342-3p inhibitor. These findings indicated \nthat miR-342-3p targeted to 3′UTR of IER3 and nega\n-\ntively regulated IER3 expression. We further investigated \nthe role of LncRNA H19 over-expression in miR-342-3p \nand IER3 expression in CD4+ T cells. We found that \nLenti-H19 significantly decreased miR-342-3p expres\n-\nsion, and miR-342-3p mimic reversed the inhibition \neffect of Lenti-H19. Lenti-H19 increased IER3 protein \nlevel, and miR-342-3p mimic reversed the promotion \neffect of Lenti-H19 (Fig. 4b).\nLncRNA H19 over‑expression suppressed Th17 \ndifferentiation and ESC proliferation through miR‑342‑3p/\nIER3\nWe went on to investigate the signaling pathway of \nLncRNA H19 in the regulation of Th17 differentiation \nand ESC proliferation. CD4+  T cells in NC and miR-\n342-3p groups were under an induced Th17 polarizing \ncondition. miR-342-3p inhibitor significantly decreased \nIL-17 level, reduced the percentage of Th17 cells/CD4+ \nFig. 4 LncRNA H19 over-expression regulated miR-342-3p/IER3 \nexpression. a Bioinformatics software predicted the miR-342-3p \ntargeted to 3′UTR of IER3. miR-342-3p mimic or inhibitor were \ntransfected into HEK293 cells to detect the luciferase activity. IER3 \nmRNA and protein levels were detected by qRT-PCR or Western \nblot after miR-342-3p mimic or inhibitor treatment. b CD4+ T \ncells were divided into lenti-NC, lenti-H19, lenti-H19 + pre-NC, \nlenti-H19 + miR-342-3p mimic groups. miR-342-3p expression and \nIER3 protein level were detected by qRT-PCR and Western blot. \n*p < 0.05, compared with pre-NC or lenti-NC; \n#p < 0.05, compared \nwith lenti-H19 + pre-NC\n\nPage 7 of 10\nLiu et al. Cell Biosci            (2019) 9:84 \nT cells, and suppressed ESC viability (Fig.  5a). CD4+ \nT cells in Lenti-NC, Lenti-H19, Lenti-H19 + pre-NC, \nLenti-H19 + miR-342-3p mimic groups were also under \nan induced Th17 polarizing condition. Then, CD4+  T \ncells were co-cultured with ESCs for 48 h. MTT assay \nand resazurin assay showed that Lenti-H19 signifi -\ncantly suppressed ESC viability, and miR-342-3p mimic \nreversed the inhibition effect of H19 over-expression \n(Fig. 5b). In addition, Lenti-H19 significantly decreased \nIL-17 level, reduced the percentage of Th17 cells/CD4+ \nT cells, and down-regulated TH17 cell marker RORγt \nexpression, and miR-342-3p mimic reversed the inhibi\n-\ntion effect of H19 over-expression (Fig. 5 c).\nLncRNA H19 over‑expression inhibited the ectopic \ngrowth of endometriosis‑like lesions in the nude mouse \nendometriosis model\nStudies have reported that Th17 differentiation pro -\nmoted the proliferation and invasion of ESC, thus to \naccelerate the growth and implantation of the endome -\ntriosis-like lesions [5 ]. At the 14th day after the estab -\nlishment of the nude mouse endometriosis model, the \nnude mice were sacrificed and the ectopic lesions were \nobtained. We observed the weight of endometriosis-\nlike lesions from nude mouse endometriosis model \nwas increased in EMS + Th17 group, and H19 over-\nexpression decreased the weight of endometriosis-\nlike lesions compare to EMS + Lenti-NC-Th17 group \n(Fig.  6a). LncRNA H19 was down-regulated in PF of \nEMS + Th17 group, and H19 over-expression increased \nLncRNA H19 expression compare to EMS + Lenti-\nNC-Th17 group (Fig.  6b). miR-342-3p expression was \nFig. 5 LncRNA H19 over-expression suppressed Th17 differentiation and ESC proliferation through miR-342-3p/IER3. a miR-342-3p inhibitor \nsuppressed IL-17 level, the percentage of Th17 cells/CD4+ T cells, and ESC viability. b CD4+ T cells were divided into lenti-NC, lenti-H19, \nlenti-H19 + pre-NC, lenti-H19 + miR-342-3p mimic groups. Then, CD4+ T cells were co-cultured with ESC for 48 h. The viability of ESC was detected \nby MTT assay and resazurin assay. c IL-17 level was detected using ELISA. The percentage of Th17 cells/CD4+ T cells was detected using flow \ncytometry. TH17 cell marker RORγt expression was detected by western blot. *p < 0.05, compared with NC or lenti-NC; \n#p < 0.05, compared with \nlenti-H19 + pre-NC\n\nPage 8 of 10Liu et al. Cell Biosci            (2019) 9:84 \nup-regulated in PF of EMS + Th17 group, and H19 \nover-expression down-regulated miR-342-3p expres -\nsion compare to EMS + Lenti-NC-Th17 group (Fig.  6b). \nProtein level of IER3 was down-regulated in PF of \nEMS + Th17 group, and H19 over-expression up-reg -\nulated IER3 level compare to EMS + Lenti-NC-Th17 \ngroup (Fig. 6 c).\nDiscussion\nThis study investigates the role of LncRNA H19 in Th17 \ncell differentiation and ESC proliferation, and its interac\n-\ntion with miR-342-3p/IER3 signaling pathway in EMS. \nThe data showed that patients with EMS had decreased \nLncRNA H19 and IER3 expressions, and increased \nmiR-342-3p expression and Th17 cells/CD4+ T cells \npercentage compared with those without EMS. Under \nTh17 polarizing condition, we found LncRNA H19 and \nIER3 expressions were down-regulated, and miR-342-3p \nexpression and IL-17 secretion were up-regulated com\n-\npared with control group. We showed that LncRNA \nH19 over-expression could decrease IL-17 secretion, \nsuppress Th17 differentiation and inhibit ESC prolifera\n-\ntion through inhibiting miR-342-3p. In vivo experiments \nstrongly indicated a suppression effect of H19 over-\nexpression on Th17 differentiation and it inhibition on \nthe growth of endometriosis-like lesions.\nEMS is characterized by the presence of ectopic endo\n-\nmetrium that causes pain, infertility and lesion progres -\nsion. The pain can be controlled by medical or surgical \ntreatment [21]. However, lesions cannot be eradicated \nFig. 6 LncRNA H19 over-expression inhibited the ectopic growth in the nude mouse endometriosis model. The nude mouse endometriosis \nmodel was established. CD4+ T cells were transfected with LncRNA H19 over-expression lentiviral vectors and induced Th17 polarizing, \nthen intraperitoneally injected into the nude mouse endometriosis model. Nude mice were divided into EMS, EMS + Th17, EMS + lenti-Th17, \nEMS + lenti-H19-Th17 groups, with six mice in each group. a The weight of endometriosis-like lesions from nude mouse endometriosis model \nwas increased in EMS + Th17 group, and H19 over-expression reversed the promotion effect of EMS + Th17. b Peritoneal fluid (PF) was collected \nto detect H19 and miR-342-3p expressions. LncRNA H19 was down-regulated in PF of EMS + Th17 group, and H19 over-expression reversed the \ninhibition effect of EMS + Th17. miR-342-3p was up-regulated in PF of EMS + Th17 group, and H19 over-expression reversed the promotion effect of \nEMS + Th17. c IER3 was down-regulated in PF of EMS + Th17 group, and H19 over-expression reversed the inhibition effect of EMS + Th17. *p < 0.05, \ncompared with EMS; #p < 0.05, compared with EMS + lenti-NC + Th17\n\nPage 9 of 10\nLiu et al. Cell Biosci            (2019) 9:84 \ndespite the pain can be managed by the pharmacologi -\ncal inhibition of ovulation and menstruation, and the \nbenefit from surgery is often temporary [22]. The recur -\nrence of symptoms and lesion is between 40 and 50% at \na 5-year follow-up period [23]. Hence, it is meaningful \nto find effective treatment for EMS. The balance of Th17 \nand regulatory T cells is important in many immuno\n-\nlogical pathologies, and it has been reported that IL-17A \nproduced by Th17 cells was highly expressed in the EMS \nlesion and could contribute to disease progression in trig\n-\ngering proinflammatory cytokines and angiogenic growth \nfactors [24]. Recent study found that in severe EMS, \nthe percentage of Th17 cells in PF was higher than that \nof early (I/II stage) EMS, which indicated the increase \nof Th17 percentage in peritoneal fluid was related with \nthe severity of EMS [24]. In this study, we showed that \nthe percentage of Th17 cells was increased in PFMC of \npatients with EMS, which was consistent with previous \nreport [25].\nNumerous studies have reported that miRNAs could \nregulate Th17 cell differentiation through binding to the \n3′-untranslated region (UTR) of the target genes [26, 27]. \nIn this study, we observed miR-342-3p and IER3 were \nabnormally expressed in PFMC of patients with EMS or \nin Th17 polarizing conditions, and miR-342-3p knock\n-\ndown suppressed IL-17 expression, the percentage of \nTh17 cells and ESC proliferation. Moreover, we first con\n-\nfirmed miR-342-3p could target to IER3 and negatively \nregulate IER3 expression.\nA number of studies have suggested that LncRNAs \ncan be abnormally expressed in EMS, and play roles \nin regulating stromal cell growth or estrogen recep\n-\ntor expression, which suggested that LncRNAs can be \nnew biomarkers or novel therapeutic targets of EMS \n[28, 29]. Ghazal et  al. showed a reduced expression \nof LncRNA H19 in the endometrium of women with \nEMS, and showed LncRNA H19 knockdown or over-\nexpression negatively or positively affect ESCs pro\n-\nliferation via regulating Let-7/Igf1r expression [17]. \nIn the present study, we showed LncRNA H19 was \ndown-regulated in PFMC of patients with EMS or in \nTh17 polarizing conditions, and proved LncRNA H19 \nover-expression inhibited IL-17 expression and the per\n-\ncentage of Th17 cells by regulating miR-342-3p/IER3. \nBesides, LncRNA H19 over-expression suppressed the \ngrowth of Th17 cell differentiation-induced endometri\n-\nosis-like lesions.\nIn conclusion, this study demonstrated that LncRNA \nH19 was down-regulated in PFMC of patients with EMS \nor under Th17 polarizing conditions, and LncRNA H19 \nover-expression suppressed Th17 cell differentiation and \nESCs proliferation through miR-342-3p/IER3 pathway.\nSupplementary information\nSupplementary information accompanies this paper at https ://doi.\norg/10.1186/s1357 8-019-0346-3.\nAdditional file 1: Figure S1. A. The percentage of Th17 cells/CD4+ T \ncells and Th17 cells/naïve CD4+ T cells in control group and induced \ngroup was detected by flow cytometry. B. IER3 protein level in PFMC from \ncontrol group and EMS group was detected by western blot. *p < 0.05, \ncompared with control.\nAcknowledgements\nNot applicable.\nAuthors’ contributions\nZL and LL put forward the concept of the study, designed the study, prepared \nthe manuscript and contributed to the statistical analysis. MC and RG contrib-\nuted to the data acquisition. JG and YZ contributed to the quality control of \ndata and algorithms. CT analyzed the data and interpretation. XH edited the \nmanuscript. YZ revised the manuscript. LH put forward the concept of the \nstudy, contributed to the data analysis and interpretation and reviewed the \nmanuscript. All authors read and approved the final manuscript.\nFunding\nThis study was supported by the Science and Technology Colleges Innovation \nTeam Support Program of Henan Province (No. 18IRTSTHN024), the Science \nand Technology Planning Project of Henan Province co-established by the \nprovince and the ministry (No. 201701002), and the National Natural Science \nFoundation of China (No. U1604172).\nAvailability of data and materials\nNot applicable\nEthics approval and consent to participate\nThe protocols were approved by the Ethics Committee of The First Affiliated \nHospital of Zhengzhou University. All patients signed informed consent.\nConsent for publication\nThe study was undertaken with the patient’s consent.\nCompeting interests\nThe authors declare that they have no competing interests.\nReceived: 4 January 2019   Accepted: 25 September 2019\nReferences\n 1. Taylor H, Osteen K, Bruner-Tran K, Lockwood C, Krikun G, Sokalska \nA, Duleba A. 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Genome-wide microarray analysis of \nlong non-coding RNAs in eutopic secretory endometrium with endome-\ntriosis. Cell Physiol Biochem. 2015;37:2231–45.\nPublisher’s Note\nSpringer Nature remains neutral with regard to jurisdictional claims in pub-\nlished maps and institutional affiliations.","source_license":"CC0","license_restricted":false}