Increased HERV-E clone 4–1 expression contributes to DNA hypomethylation and IL-17 release from CD4+ T cells via miR-302d/MBD2 in systemic lupus erythematosus
Identifieur interne : 000055 ( Pmc/Corpus ); précédent : 000054; suivant : 000056Increased HERV-E clone 4–1 expression contributes to DNA hypomethylation and IL-17 release from CD4+ T cells via miR-302d/MBD2 in systemic lupus erythematosus
Auteurs : Xin Wang ; Chaoshuai Zhao ; Chengzhong Zhang ; Xingyu Mei ; Jun Song ; Yue Sun ; Zhouwei Wu ; Weimin ShiSource :
- Cell Communication and Signaling : CCS [ 1478-811X ] ; 2019.
Abstract
Increased human endogenous retroviruses E clone 4–1 (HERV-E clone 4–1) mRNA expression is observed in systemic lupus erythematosus (SLE) patients and associates with the disease activity. In this study, we want to further investigate the mechanism of HERV-E clone 4–1 mRNA upregulation and its roles in SLE progression.
CD4+ T cells were isolated from venous blood of SLE patients or healthy controls and qRT-PCR was used to detect HERV-E clone 4–1 mRNA expression. We then investigated the regulation of Nuclear factor of activated T cells 1 (NFAT1) and Estrogen receptor-α (ER-α) on HERV-E clone 4–1 transcription and the functions of HERV-E clone 4–1 3′ long terminal repeat (LTR) on DNA hypomethylation and IL-17 release.
We found HERV-E clone 4–1 mRNA expression was upregulated in CD4+ T cells from SLE patients and positively correlated with SLE disease activity. This is associated with the activation of Ca2+/calcineurin (CaN)/NFAT1 and E2/ER-α signaling pathway and DNA hypomethylation of HERV-E clone 4–1 5’LTR. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE through miR-302d/Methyl-CpG binding domain protein 2 (MBD2)/DNA hypomethylation and IL-17 signaling via its 3’LTR.
HERV-E clone 4–1 mRNA upregulation is due to the abnormal inflammation/immune/methylation status of SLE and it could act as a potential biomarker for diagnosis of SLE. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE via its 3’LTR and the signaling pathways it involved in may be potential therapeutic targets of SLE.
The online version of this article (10.1186/s12964-019-0416-5) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/s12964-019-0416-5
PubMed: 31412880
PubMed Central: 6694475
Links to Exploration step
PMC:6694475Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Increased HERV-E clone 4–1 expression contributes to DNA hypomethylation and IL-17 release from CD4<sup>+</sup> T cells via miR-302d/MBD2 in systemic lupus erythematosus</title><author><name sortKey="Wang, Xin" sort="Wang, Xin" uniqKey="Wang X" first="Xin" last="Wang">Xin Wang</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Zhao, Chaoshuai" sort="Zhao, Chaoshuai" uniqKey="Zhao C" first="Chaoshuai" last="Zhao">Chaoshuai Zhao</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Zhang, Chengzhong" sort="Zhang, Chengzhong" uniqKey="Zhang C" first="Chengzhong" last="Zhang">Chengzhong Zhang</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Mei, Xingyu" sort="Mei, Xingyu" uniqKey="Mei X" first="Xingyu" last="Mei">Xingyu Mei</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Song, Jun" sort="Song, Jun" uniqKey="Song J" first="Jun" last="Song">Jun Song</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Sun, Yue" sort="Sun, Yue" uniqKey="Sun Y" first="Yue" last="Sun">Yue Sun</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Wu, Zhouwei" sort="Wu, Zhouwei" uniqKey="Wu Z" first="Zhouwei" last="Wu">Zhouwei Wu</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Shi, Weimin" sort="Shi, Weimin" uniqKey="Shi W" first="Weimin" last="Shi">Weimin Shi</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31412880</idno><idno type="pmc">6694475</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6694475</idno><idno type="RBID">PMC:6694475</idno><idno type="doi">10.1186/s12964-019-0416-5</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000055</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000055</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Increased HERV-E clone 4–1 expression contributes to DNA hypomethylation and IL-17 release from CD4<sup>+</sup> T cells via miR-302d/MBD2 in systemic lupus erythematosus</title><author><name sortKey="Wang, Xin" sort="Wang, Xin" uniqKey="Wang X" first="Xin" last="Wang">Xin Wang</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Zhao, Chaoshuai" sort="Zhao, Chaoshuai" uniqKey="Zhao C" first="Chaoshuai" last="Zhao">Chaoshuai Zhao</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Zhang, Chengzhong" sort="Zhang, Chengzhong" uniqKey="Zhang C" first="Chengzhong" last="Zhang">Chengzhong Zhang</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Mei, Xingyu" sort="Mei, Xingyu" uniqKey="Mei X" first="Xingyu" last="Mei">Xingyu Mei</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Song, Jun" sort="Song, Jun" uniqKey="Song J" first="Jun" last="Song">Jun Song</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Sun, Yue" sort="Sun, Yue" uniqKey="Sun Y" first="Yue" last="Sun">Yue Sun</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Wu, Zhouwei" sort="Wu, Zhouwei" uniqKey="Wu Z" first="Zhouwei" last="Wu">Zhouwei Wu</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author><author><name sortKey="Shi, Weimin" sort="Shi, Weimin" uniqKey="Shi W" first="Weimin" last="Shi">Weimin Shi</name><affiliation><nlm:aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</nlm:aff></affiliation></author></analytic><series><title level="j">Cell Communication and Signaling : CCS</title><idno type="eISSN">1478-811X</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p id="Par1">Increased human endogenous retroviruses E clone 4–1 (HERV-E clone 4–1) mRNA expression is observed in systemic lupus erythematosus (SLE) patients and associates with the disease activity. In this study, we want to further investigate the mechanism of HERV-E clone 4–1 mRNA upregulation and its roles in SLE progression.</p></sec><sec><title>Methods</title><p id="Par2">CD4<sup>+</sup> T cells were isolated from venous blood of SLE patients or healthy controls and qRT-PCR was used to detect HERV-E clone 4–1 mRNA expression. We then investigated the regulation of Nuclear factor of activated T cells 1 (NFAT1) and Estrogen receptor-α (ER-α) on HERV-E clone 4–1 transcription and the functions of HERV-E clone 4–1 3′ long terminal repeat (LTR) on DNA hypomethylation and IL-17 release.</p></sec><sec><title>Results</title><p id="Par3">We found HERV-E clone 4–1 mRNA expression was upregulated in CD4<sup>+</sup> T cells from SLE patients and positively correlated with SLE disease activity. This is associated with the activation of Ca<sup>2+</sup>/calcineurin (CaN)/NFAT1 and E2/ER-α signaling pathway and DNA hypomethylation of HERV-E clone 4–1 5’LTR. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE through miR-302d/Methyl-CpG binding domain protein 2 (MBD2)/DNA hypomethylation and IL-17 signaling via its 3’LTR.</p></sec><sec><title>Conclusions</title><p id="Par4">HERV-E clone 4–1 mRNA upregulation is due to the abnormal inflammation/immune/methylation status of SLE and it could act as a potential biomarker for diagnosis of SLE. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE via its 3’LTR and the signaling pathways it involved in may be potential therapeutic targets of SLE.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (10.1186/s12964-019-0416-5) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Yin, Y" uniqKey="Yin Y">Y Yin</name></author><author><name sortKey="Choi, S" uniqKey="Choi S">S Choi</name></author><author><name sortKey="Xu, Z" uniqKey="Xu Z">Z Xu</name></author><author><name sortKey="Perry, D" uniqKey="Perry D">D Perry</name></author><author><name sortKey="Seay, H" uniqKey="Seay H">H Seay</name></author><author><name sortKey="Croker, B" uniqKey="Croker B">B Croker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kaul, A" uniqKey="Kaul A">A 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Cell Commun Signal</journal-id><journal-id journal-id-type="iso-abbrev">Cell Commun. Signal</journal-id><journal-title-group><journal-title>Cell Communication and Signaling : CCS</journal-title></journal-title-group><issn pub-type="epub">1478-811X</issn><publisher><publisher-name>BioMed Central</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31412880</article-id><article-id pub-id-type="pmc">6694475</article-id><article-id pub-id-type="publisher-id">416</article-id><article-id pub-id-type="doi">10.1186/s12964-019-0416-5</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research</subject></subj-group></article-categories><title-group><article-title>Increased HERV-E clone 4–1 expression contributes to DNA hypomethylation and IL-17 release from CD4<sup>+</sup> T cells via miR-302d/MBD2 in systemic lupus erythematosus</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes"><name><surname>Wang</surname><given-names>Xin</given-names></name><address><email>739976260@qq.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Zhao</surname><given-names>Chaoshuai</given-names></name><address><email>l258989773@sina.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Zhang</surname><given-names>Chengzhong</given-names></name><address><email>a137748829871@126.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Mei</surname><given-names>Xingyu</given-names></name><address><email>kaiyeggut@163.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Song</surname><given-names>Jun</given-names></name><address><email>1182505137@qq.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Sun</surname><given-names>Yue</given-names></name><address><email>w7683019761@163.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Wu</surname><given-names>Zhouwei</given-names></name><address><phone>8602163240090</phone><email>zhouwei.wu@shgh.cn</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0003-2407-2204</contrib-id><name><surname>Shi</surname><given-names>Weimin</given-names></name><address><phone>8602163240090</phone><email>weiminshisjtu@163.com</email></address><xref ref-type="aff" rid="Aff1"></xref></contrib><aff id="Aff1">Department of Dermatology, Shanghai General Hospital, Shanghai Jiaotong University School of Medicine, 100 Haining Road, Shanghai, 200080 China</aff></contrib-group><pub-date pub-type="epub"><day>14</day><month>8</month><year>2019</year></pub-date><pub-date pub-type="pmc-release"><day>14</day><month>8</month><year>2019</year></pub-date><pub-date pub-type="collection"><year>2019</year></pub-date><volume>17</volume><elocation-id>94</elocation-id><history><date date-type="received"><day>23</day><month>5</month><year>2019</year></date><date date-type="accepted"><day>6</day><month>8</month><year>2019</year></date></history><permissions><copyright-statement>© The Author(s). 2019</copyright-statement><license license-type="OpenAccess"><license-p><bold>Open Access</bold>This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">http://creativecommons.org/publicdomain/zero/1.0/</ext-link>) applies to the data made available in this article, unless otherwise stated.</license-p></license></permissions><abstract id="Abs1"><sec><title>Background</title><p id="Par1">Increased human endogenous retroviruses E clone 4–1 (HERV-E clone 4–1) mRNA expression is observed in systemic lupus erythematosus (SLE) patients and associates with the disease activity. In this study, we want to further investigate the mechanism of HERV-E clone 4–1 mRNA upregulation and its roles in SLE progression.</p></sec><sec><title>Methods</title><p id="Par2">CD4<sup>+</sup> T cells were isolated from venous blood of SLE patients or healthy controls and qRT-PCR was used to detect HERV-E clone 4–1 mRNA expression. We then investigated the regulation of Nuclear factor of activated T cells 1 (NFAT1) and Estrogen receptor-α (ER-α) on HERV-E clone 4–1 transcription and the functions of HERV-E clone 4–1 3′ long terminal repeat (LTR) on DNA hypomethylation and IL-17 release.</p></sec><sec><title>Results</title><p id="Par3">We found HERV-E clone 4–1 mRNA expression was upregulated in CD4<sup>+</sup> T cells from SLE patients and positively correlated with SLE disease activity. This is associated with the activation of Ca<sup>2+</sup>/calcineurin (CaN)/NFAT1 and E2/ER-α signaling pathway and DNA hypomethylation of HERV-E clone 4–1 5’LTR. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE through miR-302d/Methyl-CpG binding domain protein 2 (MBD2)/DNA hypomethylation and IL-17 signaling via its 3’LTR.</p></sec><sec><title>Conclusions</title><p id="Par4">HERV-E clone 4–1 mRNA upregulation is due to the abnormal inflammation/immune/methylation status of SLE and it could act as a potential biomarker for diagnosis of SLE. HERV-E clone 4–1 also takes part in disease pathogenesis of SLE via its 3’LTR and the signaling pathways it involved in may be potential therapeutic targets of SLE.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (10.1186/s12964-019-0416-5) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd><italic>HERV-E clone 4–1</italic></kwd><kwd>Systemic lupus erythematosus</kwd><kwd>Transcription factors</kwd><kwd>DNA hypomethylation</kwd><kwd><italic>miR-302d</italic></kwd><kwd><italic>MBD2</italic></kwd></kwd-group><funding-group><award-group><funding-source><institution>National Natural Science Foundation of China</institution></funding-source><award-id>81573031</award-id><principal-award-recipient><name><surname>Shi</surname><given-names>Weimin</given-names></name></principal-award-recipient></award-group></funding-group><funding-group><award-group><funding-source><institution>National Natural Science Foundation of China </institution></funding-source><award-id>81773310</award-id><principal-award-recipient><name><surname>Wu</surname><given-names>Zhouwei</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2019</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Background</title><p id="Par26">Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4<sup>+</sup> T cells play an important role [<xref ref-type="bibr" rid="CR1">1</xref>]. Genetic interactions with environmental factors, particularly ultraviolet light exposure, infection and hormonal factors, might initiate the disease, resulting in immune dysregulation at the level of cytokines, T cells, B cells and macrophages [<xref ref-type="bibr" rid="CR2">2</xref>].</p><p id="Par27">Human endogenous retroviruses (HERV) are descendants of occasional germline invasion by exogenous retroviruses which occupy as much as 8% of the human genome [<xref ref-type="bibr" rid="CR3">3</xref>]. <italic>HERV-E clone 4–1</italic> is inserted in the short arm of chromosome 19 at position 19p12 upstream of the <italic>ZNF66</italic> gene locus and in the antisense orientation. This full-length HERV-E clone 4–1 is considered to be an LTR2C prototype containing 5′ and 3′ LTR elements that are 95.5% identical and encompass gag, pol and env genes (GenBank: M10976, Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Figure S1) [<xref ref-type="bibr" rid="CR4">4</xref>]. Enhanced expression of mRNA from <italic>HERV-E clone 4–1</italic> was reported in SLE than healthy controls (HCs) [<xref ref-type="bibr" rid="CR5">5</xref>, <xref ref-type="bibr" rid="CR6">6</xref>], and our former study demonstrated that <italic>HERV-E clone 4–1</italic> mRNA expression was increased in SLE patients, and the expression level of <italic>HERV-E clone 4–1</italic> was associated with SLE disease activity index (SLEDAI) [<xref ref-type="bibr" rid="CR7">7</xref>]. <italic>HERV-E clone 4–1</italic> 5’LTR/LTR2C was hypomethylated in CD4<sup>+</sup> T cells from SLE patients [<xref ref-type="bibr" rid="CR7">7</xref>–<xref ref-type="bibr" rid="CR9">9</xref>] which might have close relationship with its expression.</p><p id="Par28">In this study, we sought to further investigate the mechanism of <italic>HERV-E clone 4–1</italic> mRNA upregulation and its roles in SLE progression, and to estimate the potential value of <italic>HERV-E clone 4–1</italic> in acting as a biomarker and therapeutic target for SLE.</p></sec><sec id="Sec2"><title>Methods</title><sec id="Sec3"><title>Ethics and selection of patients</title><p id="Par29">This research was approved by the Institutional Research Ethics Committee of Shanghai General Hospital and abided by the ethical guidelines of the Declaration of Helsinki. All the patients involved in this study were adult and written informed consents were obtained from all the patients. All patients with SLE were diagnosed in accordance with the 1997 ACR revised criteria for classification of SLE. Disease activity was assessed using the SLE disease activity index (SLEDAI), and active disease was defined as an SLEDAI score ≥ 5. Age- and sex-matched healthy controls were recruited from the medical staff at Shanghai General Hospital.</p></sec><sec id="Sec4"><title>Isolation, culture and treatment of CD4<sup>+</sup> T cells</title><p id="Par30">Peripheral blood mononuclear cells (PBMC) were isolated from venous blood of SLE patients or healthy controls using Ficoll-paque density gradient centrifugation. Purified CD4<sup>+</sup> T cells were negatively isolated from PBMCs by CD4<sup>+</sup> T-cell isolation kits (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s protocol. CD4<sup>+</sup> T cell purity was routinely > 90% as verified through flow cytometry. The cells were then cultured in Xvivo 15 medium (Lonza, Walkersville, MD, USA) supplemented with 10% human AB serum (Valley Biomedical, Winchester, VA, USA) at 37 °C with 5% CO<sub>2</sub>. The treatments of the cells were: TNF-α (HY-P7058, MedChemExpress, NJ, USA), 10 ng/ml, 24 h; IL-6 (HY-P7044, MedChemExpress), 10 ng/ml, 24 h; 17β-estradiol (estradiol/E2) (HY-B0141, MedChemExpress), 100 nmol/L, 24 h; Lipopolysaccharides (LPS) (L8880, Solarbio, Beijing, China), 100 ng/ml, 24 h; ultraviolet B (UVB), 50 mJ/cm2 [<xref ref-type="bibr" rid="CR10">10</xref>]; hydroxychloroquine sulfate (HCQ sulfate) (HY-B1370, MedChemExpress), 6 μg/ml, 24 h; 5-Azacytidine (5-aza C) (HY-10586, MedChemExpress), 1 mM, 24 h; prednisolone (HY-17463, MedChemExpress), 10 ng/ml, 24 h; AZD9496 (HY-12870, MedChemExpress), 5 nM, 24 h.</p></sec><sec id="Sec5"><title>Quantitative reverse transcription-PCR (qRT-PCR)</title><p id="Par31">Total RNAs of cells were extracted using Trizol (Invitrogen) according to the instructions provided by the manufacturer. Reverse transcription was performed using the Primescript RT Master Mix (Takara, Otsu, Japan), and cDNA was amplified using SYBR-Green Premix (Takara). The expression of <italic>HERV-E clone 4–1 gag</italic> was normalized to the expressions of <italic>GAPDH</italic>. The data were analyzed by delta Ct method. Primers of <italic>HERV-E clone 4–1</italic> gag used in this study were imported from other published articles [<xref ref-type="bibr" rid="CR5">5</xref>–<xref ref-type="bibr" rid="CR7">7</xref>] and the primers were, F: 5′-CACATGGTGGAGAGTCGTGTTT-3′ and R: 5′-GCTTGCGGCTTTTCAGTATAGG-3′; <italic>GAPDH</italic>, F: 5′-GGAGTCCACTGGCGTCTTC-3′ and R: 5′-GCTGATGATCTTGAGGCTGTTG-3′. Primers for <italic>HERV-E clone 4–1</italic> 3’LTR were, F: 5′-TCGCCACTTCTCCTGTTGTC-3′ and R: 5′-TATTCGGCCGGGATCATTGG-3′.</p></sec><sec id="Sec6"><title>Oligonucleotide, plasmids and transfection</title><p id="Par32">SiRNA, <italic>miR-302d</italic> mimics and corresponding negative controls were transfected by Hiperfect transfection reagent (Qiagen, Valencia, CA, USA) and plasmids were transfected by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) into cells. <italic>Nuclear factor of activated T cells 1</italic> (<italic>NFAT1</italic>) siRNA and <italic>Estrogen receptor-α</italic> (<italic>ER-α</italic>) siRNA were obtained from Santa Cruz Biotechnology (sc-36,055, sc-29,305, Santa Cruz, CA, USA). The 3’LTR of <italic>HERV-E clone 4–1</italic> were cloned into pcDNA 3.1 plasmid and the recombinant plasmid was transfected into cells to obtain the 3’LTR mRNA overexpression.</p></sec><sec id="Sec7"><title>Western blot analysis</title><p id="Par33">Cells were lysed using radioimmunoprecipitation (RIPA) lysis buffer (Beyotime, Shanghai, China). Protein concentrations were detected using bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Antibodies used in the assays were <italic>NFAT1</italic> antibody (ab2722, Abcam, Cambridge, UK), <italic>ER-α</italic> antibody (#8644, Cell Signaling Technology) and GAPDH antibody (#5174, Cell Signaling Technology), <italic>IRF9</italic> antibody (#76684, Cell Signaling Technology), <italic>Methyl-CpG binding domain protein 2</italic> (<italic>MBD2</italic>) antibody (ab38646, Abcam) and IL-17 antibody (ab77171, Abcam).</p></sec><sec id="Sec8"><title>Luciferase assay</title><p id="Par34">An NFAT luciferase reporter plasmid (pNFAT-Luc) containing <italic>NFAT1</italic> binding promoter elements was used to detect the <italic>NFAT1</italic> transcriptional activity. CD4<sup>+</sup> T cells were co-transfected with a mixture of 300 ng pNFAT-Luc reporter and 5 ng pRL-TK Renilla luciferase reporter. After different treatment, the luciferase activities were measured using the Dual Luciferase Reporter assay (Promega, Madison, WI, USA). pRL-TK Renilla luciferase reporter was used to normalize the transfection efficiency.</p><p id="Par35">Full-length sequences of <italic>HERV-E clone 4–1</italic> 5′ LTR containing wild-type of <italic>NFAT1</italic> or <italic>ER-α</italic> predicted binding site was inserted into PGL3-Basic luciferase reporter vector (Promega). Mutant reporter plasmids were prepared using Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Cells were co-transfected with a mixture of 300 ng firefly luciferase reporter, 5 ng pRL-TK Renilla luciferase reporter, and <italic>NFAT1</italic> or <italic>ER-α</italic> plasmids. After 48 h of incubation, the luciferase activities were quantified using the Dual Luciferase Assay System (Promega). The sequences of 3’LTR of <italic>HERV-E clone 4–1</italic> mRNA or <italic>MBD2</italic> 3’UTR containing potential wild-type or mutant binding sites of <italic>miR-302d</italic> were constructed into pmirGLO vectors (Promega). The luciferase vectors and <italic>miR-302d</italic> mimics were transfected into CD4<sup>+</sup> cells along with pRL-TK vector. The dual-luciferase Reporter assay system (Promega) was used to detect luciferase activity. pRL-TK Renilla luciferase reporter was used to normalize the transfection efficiency.</p></sec><sec id="Sec9"><title>Chromatin immunoprecipitation (ChIP)</title><p id="Par36">ChIP assay was conducted using EZ ChIP kit (Millipore, Billerica, MA, USA) and <italic>NFAT1</italic> antibody (ab2722, Abcam) or <italic>ER-α</italic> antibody (#8644, Cell Signaling Technology) according to the instruction of the manufacturer. The primers specific to <italic>HERV-E clone 4–1</italic> 5′ LTR were: 5′-CTCCCCAACCTCCCCTTTTC-3′ and 5′-TGAGAAACATGACTGGGGGC-3′. Normal rabbit IgG (A7016, Beyotime, Shanghai, China) was used to control the nonspecific immunoprecipitation.</p></sec><sec id="Sec10"><title>DNA extraction and global methylation analysis</title><p id="Par37">Assays of DNA extraction and global methylation analysis was described in our previous study [<xref ref-type="bibr" rid="CR11">11</xref>].</p></sec><sec id="Sec11"><title>Enzyme-linked immunosorbent assay</title><p id="Par38">The concentration of IL-17 in culture supernatants were measured by Human IL-17 ELISA Kit (ab119535, Abcam) according to the manufacturer’s instructions. Optical density values were read at 450 nm using ELx800 Absorbance Microplate Reader (BioTek, VT, USA).</p></sec><sec id="Sec12"><title>Statistical analysis</title><p id="Par39">Statistical analysis was performed using the SPSS program (version 18.0; SPSS, Chicago, IL, USA). The statistical significance of differences between two groups was tested using Student’s t test. Spearman’s analysis was used to test correlation. <italic>P</italic> < 0.05 was considered as statistically significant.</p></sec></sec><sec id="Sec13"><title>Results</title><sec id="Sec14"><title><italic>HERV-E clone 4–1</italic> mRNA expression was upregulated in CD4<sup>+</sup> T cells from SLE patients</title><p id="Par40">In our former study, we found that <italic>HERV-E clone 4–1</italic> mRNA expression was higher in lupus CD4<sup>+</sup> T cells than in cells from healthy controls and the <italic>HERV-E clone 4–1</italic> mRNA expression level was positively correlated with SLE disease activity [<xref ref-type="bibr" rid="CR7">7</xref>]. To continue our study, first, we used new samples (Additional file <xref rid="MOESM1" ref-type="media">1</xref>: Table S1) to prove <italic>HERV-E clone 4–1</italic> mRNA expression was higher in SLE CD4<sup>+</sup> T cells than in cells from healthy controls using Quantitative reverse transcription-PCR (qRT-PCR) (Fig. <xref rid="Fig1" ref-type="fig">1</xref>a). We also found that <italic>HERV-E clone 4–1</italic> mRNA expression level was higher in active patients than that of inactive patients (Fig. <xref rid="Fig1" ref-type="fig">1</xref>b) and positively correlated with SLE disease activity (Fig. <xref rid="Fig1" ref-type="fig">1</xref>c). We also followed-up some patients who got oral prednisolone and hydroxychloroquine treatment and the activity of SLE changed from active to inactive. We found that the <italic>HERV-E clone 4–1</italic> mRNA expressions decreased as the SLEDAI decreased (Fig. <xref rid="Fig1" ref-type="fig">1</xref>d and e). But the <italic>HERV-E clone 4–1</italic> mRNA expressions of the inactive patients were also higher than that of HCs (Fig. <xref rid="Fig1" ref-type="fig">1</xref>f). What’s more, to assess the diagnostic value of <italic>HERV-E clone 4–1</italic> mRNA for SLE, we performed Receiver Operating Characteristic (ROC) curve analysis to differentiate SLE from HC with the relative <italic>HERV-E clone 4–1</italic> mRNA expressions of the SLE patients and healthy controls (Fig. <xref rid="Fig1" ref-type="fig">1</xref>f). The Area Under Curve (AUC) was 0.760, the 95% confidence interval (95% CI) was 0.622 to 0.897, and the best Youden’s index is 0.5. This indicated that <italic>HERV-E clone 4–1</italic> mRNA might have good diagnostic value for SLE and could act as a potential diagnostic biomarker for SLE.
<fig id="Fig1"><label>Fig. 1</label><caption><p><italic>HERV-E clone 4–1</italic> mRNA expression was upregulated in CD4<sup>+</sup> T cells from SLE patients. <bold>a</bold> and <bold>b</bold>) Relative expression of <italic>HERV-E clone 4–1</italic> mRNA in CD4<sup>+</sup> cells from SLE patients (<italic>N</italic> = 27) and healthy controls (HCs) (<italic>N</italic> = 21) or active SLE patients (<italic>N</italic> = 24) and inactive SLE patients (<italic>N</italic> = 3) compared using the unpaired Student’s t test. <bold>c</bold> Correlation between expression of <italic>HERV-E clone 4–1</italic> mRNA and SLEDAI score analyzed with Spearman’s analysis. <bold>d</bold> and <bold>e</bold> SLEDAI score and <italic>HERV-E clone 4–1</italic> mRNA expression in patients who got oral prednisolone and hydroxychloroquine treatment (<italic>N</italic> = 9); data were compared using the paired Student’s t test. <bold>f</bold> Relative expression of <italic>HERV-E clone 4–1</italic> mRNA in CD4<sup>+</sup> cells from inactive SLE patients (<italic>N</italic> = 11) and HCs (<italic>N</italic> = 21) compared using the unpaired Student’s t test. <bold>g</bold> ROC curve of relative <italic>HERV-E clone 4–1</italic> mRNA expression for differentiating SLE patients from HCs. Data were represented as mean ± SD. *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001</p></caption><graphic xlink:href="12964_2019_416_Fig1_HTML" id="MO1"></graphic></fig></p></sec><sec id="Sec15"><title><italic>NFAT1</italic> activity was increased in SLE and associated with increased <italic>HERV-E clone 4–1</italic> mRNA</title><p id="Par41">To explain why <italic>HERV-E clone 4–1</italic> mRNA was upregulated in CD4<sup>+</sup> T cells from SLE patients, we wondered if some transcription factors could promote the transcription of <italic>HERV-E clone 4–1</italic> mRNA. Since the 5′ LTR contained the transcription factor binding sites [<xref ref-type="bibr" rid="CR12">12</xref>], this region was used to predict the potential transcription factors. Using TransFac and JASPAR database, we found some transcription factors that might regulate the expression of <italic>HERV-E clone 4–1</italic> mRNA. <italic>NFAT1</italic>, which was proved to play critical roles in SLE [<xref ref-type="bibr" rid="CR13">13</xref>] caught our attention. First, full length fragment of the human Human endogenous retroviral DNA (4–1) 5′ LTR with wild type (wt) or mutant (mut) predicted <italic>NFAT1</italic> binding site was inserted into the luciferase reporter plasmid (Fig. <xref rid="Fig2" ref-type="fig">2</xref>a). Then, we use <italic>NFAT1</italic> overexpression plasmids to overexpress <italic>NFAT1</italic> and <italic>NFAT1</italic> siRNA to knockdown <italic>NFAT1</italic> (Fig. <xref rid="Fig2" ref-type="fig">2</xref>b-e). Luciferase reporter analysis showed that overexpression of <italic>NFAT1</italic> led to an increase in luciferase activity of the wt <italic>HERV-E clone 4–1</italic> 5’LTR plasmid in CD4<sup>+</sup> T cells, while mut <italic>NFAT1</italic> binding site attenuated the increase of luciferase activity (Fig. <xref rid="Fig2" ref-type="fig">2</xref>f). In addition, Chromatin immunoprecipitation (ChIP) assay clearly showed that the predicted <italic>NFAT1</italic>-binding site in <italic>HERV-E clone 4–1</italic> 5′ LTR presented the ability to bind to <italic>NFAT1</italic> protein (Fig. <xref rid="Fig2" ref-type="fig">2</xref>g). Moreover, qRT-PCR analysis showed that overexpression of <italic>NFAT1</italic> could increase the expression of <italic>HERV-E clone 4–1</italic> mRNA and knockdown of <italic>NFAT1</italic> with siRNA could decrease the expression of <italic>HERV-E clone 4–1</italic> mRNA (Fig. <xref rid="Fig2" ref-type="fig">2</xref>h and i). Then, we collected CD4+ T cells of SLE patients and HCs to detected <italic>NFAT1</italic> activity using NFAT luciferase reporter assay and <italic>HERV-E clone 4–1</italic> mRNA expression. We found <italic>NFAT1</italic> activity was upregulated in CD4+ T cells from SLE patients (Fig. <xref rid="Fig2" ref-type="fig">2</xref>j) and higher in active patients than that of inactive patients (Fig. <xref rid="Fig1" ref-type="fig">1</xref>k). What’s more, the relative <italic>NFAT1</italic> activity had strong correlation with <italic>HERV-E clone 4–1</italic> mRNA expression (Fig. <xref rid="Fig2" ref-type="fig">2</xref>l). So, these results all together suggested that <italic>NFAT1</italic> could induce <italic>HERV-E clone 4–1</italic> mRNA expression via binding to its 5′ LTR. We also detected the influence of some factors on <italic>NFAT1</italic> activity and <italic>HERV-E clone 4–1</italic> mRNA expression (Fig. <xref rid="Fig2" ref-type="fig">2</xref>m and n).
<fig id="Fig2"><label>Fig. 2</label><caption><p><italic>NFAT1</italic> activity was upregulated in CD4<sup>+</sup> T cells from SLE patients and closely associated with increased <italic>HERV-E clone 4–1</italic> mRNA expression. <bold>a</bold> Predicted wild-type (wt) binding sites and corresponding mutant (mut) sites of <italic>NFAT1</italic> on <italic>HERV-E clone 4–1</italic> 5’LTR. <bold>b</bold>-<bold>e</bold> qRT-PCR and western-blot assays showing relative <italic>NFAT1</italic> mRNA and protein expression in CD4<sup>+</sup> cells from SLE patient with <italic>NFAT1</italic> overexpression or knockdown. <bold>f</bold> Luciferase assays were performed in CD4<sup>+</sup> T cells from SLE patient transfected with wt or mut luciferase reporter. Each luciferase activity was normalized to the value obtained in the cells transfected with vector (N = 3). <bold>g</bold> ChIP assay was used to assess <italic>NFAT1</italic> binding site at <italic>HERV-E clone 4–1</italic> 5’LTR. <bold>h</bold> and <bold>i</bold>) Relative <italic>HERV-E clone 4–1</italic> mRNA expression in CD4<sup>+</sup> cells from SLE patient with <italic>NFAT1</italic> overexpression or knockdown (<italic>N</italic> = 3). <bold>j</bold> and <bold>k</bold> Relative <italic>NFAT1</italic> activity in CD4<sup>+</sup> cells from SLE patients (<italic>N</italic> = 15) and HCs (<italic>N</italic> = 13) or active SLE patients (<italic>N</italic> = 12) and inactive SLE patients (<italic>N</italic> = 3) compared using the unpaired Student’s t test. <bold>l</bold> Correlation between relative <italic>NFAT1</italic> activity and relative <italic>HERV-E clone 4–1</italic> mRNA expression analyzed with Spearman’s analysis (<italic>N</italic> = 15). <bold>m</bold> and <bold>n</bold> CD4<sup>+</sup> T cells from SLE patient were treated with TNF-α, IL-6, E2, LPS, UVB, HCQ or prednisolone in vitro. Relative <italic>NFAT1</italic> activity (L) and relative <italic>HERV-E clone 4–1</italic> mRNA expression (m) were detected. Data were represented as mean ± SD. *P < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001</p></caption><graphic xlink:href="12964_2019_416_Fig2_HTML" id="MO2"></graphic></fig></p></sec><sec id="Sec16"><title>E2 could upregulate <italic>HERV-E clone 4–1</italic> mRNA expression via <italic>ER-α</italic> in CD4<sup>+</sup> T cells from SLE patients</title><p id="Par42">When selecting the potential transcript factors that might regulate the expression of <italic>HERV-E clone 4–1</italic> mRNA, <italic>ER-α</italic>, which was the receptor of E2 drew our attention. This is because SLE has a predilection for females of child-bearing age who have relatively high estrogen level and estrogen is also a risk factor for SLE [<xref ref-type="bibr" rid="CR14">14</xref>] and HERV-E was upregulated in breast cancer and ovarian cancer [<xref ref-type="bibr" rid="CR15">15</xref>, <xref ref-type="bibr" rid="CR16">16</xref>]. Then, we further explored the role of E2 and <italic>ER-α</italic> in SLE. Accordingly, full length fragment of the human endogenous retroviral DNA (4–1) 5′ LTR with wild type (wt) or mutant (mut) predicted <italic>ER-α</italic> binding site was inserted into the luciferase reporter plasmid (Fig. <xref rid="Fig3" ref-type="fig">3</xref>a). Then, we use <italic>ER-α</italic> overexpression plasmids to overexpress <italic>ER-α</italic> and <italic>ER-α</italic> siRNA to knockdown <italic>ER-α</italic> (Fig. <xref rid="Fig3" ref-type="fig">3</xref>b-e). Luciferase reporter analysis showed that overexpression of <italic>ER-α</italic> led to an increase in luciferase activity of the wt <italic>HERV-E clone 4–1</italic> 5’LTR plasmid in CD4<sup>+</sup> T cells, while mut <italic>ER-α</italic> binding site attenuated the increase of luciferase activity (Fig. <xref rid="Fig3" ref-type="fig">3</xref>f). In addition, ChIP assay clearly showed that the predicted <italic>ER-α</italic>-binding site in <italic>HERV-E clone 4–1</italic> 5′ LTR presented the ability to bind to <italic>ER-α</italic> protein (Fig. <xref rid="Fig3" ref-type="fig">3</xref>g). Moreover, qRT-PCR analysis showed that <italic>ER-α</italic> plasmids could increase the expression of <italic>HERV-E clone 4–1</italic> mRNA while <italic>ER-α</italic> antagonist AZD9496 maleate and <italic>ER-α</italic> siRNA could decrease the expression of <italic>HERV-E clone 4–1</italic> mRNA (Fig. <xref rid="Fig3" ref-type="fig">3</xref>h-j). In addition, AZD9496 and <italic>ER-α</italic> siRNA could reverse the upregulated <italic>HERV-E clone 4–1</italic> mRNA expression induced by E2 (Fig. <xref rid="Fig3" ref-type="fig">3</xref>k and l). So, these results all together suggested that E2 could also upregulate <italic>HERV-E clone 4–1</italic> mRNA expression via <italic>ER-α</italic> in CD4<sup>+</sup> T cells from SLE patients.
<fig id="Fig3"><label>Fig. 3</label><caption><p>E2 could upregulate <italic>HERV-E clone 4–1</italic> mRNA expression via <italic>ER-α</italic> in CD4<sup>+</sup> T cells from SLE patients. <bold>a</bold> Predicted wild-type (wt) binding sites and corresponding mutant (mut) sites of <italic>ER-α</italic> on <italic>HERV-E clone 4–1</italic> 5’LTR. <bold>b</bold>-<bold>e</bold> qRT-PCR and western-blot assays showing relative <italic>ER-α</italic> mRNA and protein expression in CD4+ cells from SLE patient with <italic>ER-α</italic> overexpression or knockdown. <bold>f</bold> Luciferase assays were performed in CD4<sup>+</sup> T cells from SLE patient transfected with wt or mut luciferase reporter. Each luciferase activity was normalized to the value obtained in the cells transfected with vector. <bold>g</bold> ChIP assay was used to assess <italic>ER-α</italic> binding site at <italic>HERV-E clone 4–1</italic> 5’LTR. <bold>h</bold> Relative <italic>HERV-E clone 4–1</italic> mRNA expression in CD4+ cells from SLE patient with <italic>ER-α</italic> overexpression compared using the paired Student’s t test. <bold>i</bold> and <bold>j</bold> Relative <italic>HERV-E clone 4–1</italic> mRNA expression in CD4<sup>+</sup> cells from SLE patient with <italic>ER-α</italic> inhibition using siRNA or AZD9496 compared using the paired Student’s t test. <bold>k</bold> and <bold>l</bold> Relative <italic>HERV-E clone 4–1</italic> mRNA expression in CD4<sup>+</sup> cells from SLE patient when <italic>ER-α</italic> siRNA or AZD9496 was used after E2 treatment compared using the paired Student’s t test. Data were represented as mean ± SD, <italic>N</italic> = 3. *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001</p></caption><graphic xlink:href="12964_2019_416_Fig3_HTML" id="MO3"></graphic></fig></p></sec><sec id="Sec17"><title>DNA hypomethylation of <italic>HERV-E clone 4–1</italic> 5’LTR contributed to the increase of <italic>HERV-E clone 4–1</italic> mRNA</title><p id="Par43">In our former study, we found the <italic>HERV-E clone 4–1</italic> 5’LTR was hypomethylated in CD4<sup>+</sup> T cells from SLE patients and its methylation could be inhibited by 5-aza C [<xref ref-type="bibr" rid="CR7">7</xref>]. Here, we investigated whether this DNA hypomethylation was involved in the <italic>NFAT1</italic> or <italic>ER-α</italic> induced <italic>HERV-E clone 4–1</italic> mRNA upregulation. We found that <italic>HERV-E clone 4–1</italic> mRNA expressions were upregulated when <italic>NFAT1</italic> or <italic>ER-α</italic> was overexpressed or 5-aza C was used in CD4<sup>+</sup> T cells from SLE patients and HCs (Fig. <xref rid="Fig4" ref-type="fig">4</xref>a-d). In CD4<sup>+</sup> T cells from SLE patients and HCs, <italic>HERV-E clone 4–1</italic> mRNA expressions were higher when both <italic>NFAT1</italic> was overexpressed and 5-aza C was used than that when <italic>NFAT1</italic> was overexpressed or 5-aza C was used (Fig. <xref rid="Fig4" ref-type="fig">4</xref>a and b); accordingly, <italic>HERV-E clone 4–1</italic> mRNA expressions were higher when both <italic>ER-α</italic> was overexpressed and 5-aza C was used than that when <italic>ER-α</italic> was overexpressed or 5-aza C was used (Fig. <xref rid="Fig4" ref-type="fig">4</xref>c and d). Besides, the times of <italic>HERV-E clone 4–1</italic> mRNA upregulation were higher in CD4<sup>+</sup> T cells from SLE patients than that of HCs when <italic>NFAT1</italic> or <italic>ER-α</italic> was overexpressed (Fig. <xref rid="Fig4" ref-type="fig">4</xref>e and f), and the times of <italic>HERV-E clone 4–1</italic> mRNA upregulation were higher in CD4<sup>+</sup> T cells from HCs than that of SLE patients when 5-aza C was used (Fig. <xref rid="Fig4" ref-type="fig">4</xref>g). These results together suggested that DNA hypomethylation contributed to the upregulation of <italic>HERV-E clone 4–1</italic> mRNA induced by <italic>NFAT1</italic> and <italic>ER-α</italic>.
<fig id="Fig4"><label>Fig. 4</label><caption><p>DNA hypomethylation of <italic>HERV-E clone 4–1</italic> 5’LTR contributed to upregulation of <italic>HERV-E clone 4–1</italic> mRNA induced by <italic>NFAT1</italic> and <italic>ER-α</italic><bold>.</bold> CD4<sup>+</sup> T cells from SLE patient or HC were treated with <italic>NFAT1</italic> plasmids, <italic>ER-α</italic> plasmids, 5-aza C alone or in combination with 5-aza C in vitro. Relative <italic>HERV-E clone 4–1</italic> mRNA expression (<bold>a</bold>-<bold>d</bold>) were detected. <bold>e</bold> and <bold>f</bold> Times of <italic>HERV-E clone 4–1</italic> mRNA upregulation were compared by the Student’s t test in CD4<sup>+</sup> T cells from SLE patient and HC when <italic>NFAT1</italic> or <italic>ER-α</italic> was overexpressed. <bold>g</bold> Times of <italic>HERV-E clone 4–1</italic> mRNA upregulation were compared by the Student’s t test in CD4<sup>+</sup> T cells from SLE patient and HC when 5-aza C was used. Data were represented as mean ± SD, <italic>N</italic> = 3. *<italic>P</italic> < 0.05, **<italic>P</italic> < 0.01, ***<italic>P</italic> < 0.001</p></caption><graphic xlink:href="12964_2019_416_Fig4_HTML" id="MO4"></graphic></fig></p></sec><sec id="Sec18"><title><italic>HERV-E clone 4–1</italic> 3’LTR induced DNA hypomethylation and IL-17 release via <italic>miR-302d</italic>/<italic>MBD2</italic></title><p id="Par44">Since 3’UTRs of mRNAs were reported to act as natural miRNA sponges and could serve as competitive endogenous RNAs (ceRNAs) of other genes through sharing the common miRNAs [<xref ref-type="bibr" rid="CR17">17</xref>–<xref ref-type="bibr" rid="CR20">20</xref>]. We want to explore whether the 3’LTR of <italic>HERV-E clone 4–1</italic> mRNA could act as a miRNA sponge and act ceRNAs of other genes. Through programs based on <ext-link ext-link-type="uri" xlink:href="http://microrna.org">microRNA.org</ext-link> and Targetscan, we found that there was a potential binding site of <italic>miR-302d</italic> in the 3’LTR of <italic>HERV-E clone 4–1</italic> mRNA (Fig. <xref rid="Fig5" ref-type="fig">5</xref>a). Then, we performed luciferase reporter assays to determine this interaction. Luciferase assay showed that <italic>miR-302d</italic> mimics could decrease the luciferase activity of reporter containing wt 3’LTR of <italic>HERV-E clone 4–1</italic> while mut binding site attenuated the increase of luciferase activity (Fig. <xref rid="Fig5" ref-type="fig">5</xref>b). This suggested that 3’LTR of <italic>HERV-E clone 4–1</italic> could bind to <italic>miR-302d</italic> and act as a sponge for <italic>miR-302d</italic>. We also found <italic>MBD2</italic> was another potential target of <italic>miR-302d</italic> (Fig. <xref rid="Fig5" ref-type="fig">5</xref>a) and verified the interaction between <italic>MBD2</italic> 3’UTR and <italic>miR-302d</italic> using luciferase assay (Fig. <xref rid="Fig5" ref-type="fig">5</xref>c). Then, we found that overexpression of 3’LTR of <italic>HERV-E clone 4–1</italic> (Fig. <xref rid="Fig5" ref-type="fig">5</xref>d) increased the protein levels of <italic>MBD2</italic> and <italic>miR-302d</italic> mimics could rescue the increase of <italic>MBD2</italic> protein by the 3’LTR (Fig. <xref rid="Fig5" ref-type="fig">5</xref>e). These results suggested that <italic>HERV-E clone 4–1</italic> acts as a ceRNA of <italic>MBD2</italic> to positively regulate <italic>MBD2</italic> expression in 3’LTR and <italic>miR-302d</italic> dependent manners. We also detected the expression of <italic>IRF-9</italic> which was a proved target of <italic>miR-302d</italic> in SLE [<xref ref-type="bibr" rid="CR21">21</xref>] and found that overexpression of 3’LTR of <italic>HERV-E clone 4–1</italic> increased the protein levels of <italic>IRF9</italic> and <italic>miR-302d</italic> mimics could rescue the increase of IRF9 protein by the 3’LTR (Fig. <xref rid="Fig5" ref-type="fig">5</xref>e).
<fig id="Fig5"><label>Fig. 5</label><caption><p><italic>HERV-E clone 4–1</italic> 3’LTR induced DNA hypomethylation and IL-17 release via <italic>miR-302d</italic>/<italic>MBD2</italic> in CD4+ T cells of SLE. <bold>a</bold> Predicted binding sites of <italic>miR-302d</italic> in the 3’LTR of <italic>HERV-E clone 4–1</italic> mRNA and <italic>MBD2</italic> 3’UTR. <bold>b</bold> and <bold>c</bold> Effects of <italic>miR-302d</italic> on the expression of luciferase reporter genes containing <italic>HERV-E clone 4–1</italic> 3’LTR or <italic>MBD2</italic> 3’UTR. Luciferase activity was normalized to the value obtained in cells transfected with NC oligonucleotides. <bold>d</bold> Relative expression of <italic>HERV-E clone 4–1</italic> 3’LTR mRNA when CD4<sup>+</sup> T cells were transfected with <italic>HERV-E clone 4–1</italic> 3’LTR expression plasmids. <bold>e</bold> Western blot analysis of IRF9 and <italic>MBD2</italic> proteins in CD4<sup>+</sup> T cells from SLE patients transfected with <italic>HERV-E clone 4–1</italic> 3’LTR expression plasmids and/or <italic>miR-302d</italic> mimics and the corresponding negative controls. Relative global DNA methylation level (<bold>f</bold>), intracellular IL-17 level (<bold>g</bold>-<bold>i</bold>) and IL-17 level in culture supernatants (<bold>j</bold>) in CD4+ T cells from SLE patient transfected with <italic>HERV-E clone 4–1</italic> 3’LTR expression plasmids, <italic>miR-302d</italic> mimics or <italic>MBD2</italic> expression plasmids and the corresponding negative controls</p></caption><graphic xlink:href="12964_2019_416_Fig5_HTML" id="MO5"></graphic></fig></p><p id="Par45">The mRNA levels of <italic>MBD2</italic> in was increased in CD4<sup>+</sup> T cells of SLE patients and inversely correlated with global DNA methylation and positively correlated with and SLEDAI score [<xref ref-type="bibr" rid="CR22">22</xref>, <xref ref-type="bibr" rid="CR23">23</xref>]. What’s more, <italic>MBD2</italic> was found to stimulates Th17 cell differentiation and IL-17 release in other autoimmune diseases [<xref ref-type="bibr" rid="CR24">24</xref>–<xref ref-type="bibr" rid="CR26">26</xref>] and IL-17 play critical functions in the pathophysiology of SLE [<xref ref-type="bibr" rid="CR27">27</xref>, <xref ref-type="bibr" rid="CR28">28</xref>] So, <italic>MBD2</italic> might play important roles in SLE progression. Then, we intended to further study the role of <italic>HERV-E clone 4–1</italic>, <italic>miR-302d</italic> and <italic>MBD2</italic> in global DNA methylation and IL-17 expression in CD4<sup>+</sup> T cells of SLE patients. CD4+ T cells were transfected with <italic>HERV-E clone 4–1</italic> 3’LTR expression plasmids, <italic>miR-302d</italic> mimics or <italic>MBD2</italic> expression plasmids. Global DNA methylation levels, intracellular IL-17 level and IL-17 level in culture supernatants were subsequently measured. The results showed that global DNA methylation level decreased when CD4<sup>+</sup> T cells of SLE were transfected with 3’LTR expression plasmids or <italic>MBD2</italic> expression plasmids and increased when transfected with <italic>miR-302d</italic> mimics (Fig. <xref rid="Fig5" ref-type="fig">5</xref>f). Intracellular IL-17 level and IL-17 level in culture supernatants increased when CD4<sup>+</sup> T cells of SLE were transfected with 3’LTR expression plasmids or <italic>MBD2</italic> expression plasmids and decreased when transfected with <italic>miR-302d</italic> mimics (Fig. <xref rid="Fig5" ref-type="fig">5</xref>g-j). All together, these results suggested that <italic>HERV-E clone 4–1</italic> 3’LTR induce DNA hypomethylation and IL-17 release via <italic>miR-302d</italic>/<italic>MBD2</italic> in CD4<sup>+</sup> T cells of SLE.</p></sec></sec><sec id="Sec19"><title>Discussion</title><p id="Par46">Some studies had proved that <italic>HERV-E clone 4–1</italic> mRNA expression was increased in SLE patients, and the expression level of <italic>HERV-E clone 4–1</italic> was associated with SLE disease activity [<xref ref-type="bibr" rid="CR5">5</xref>–<xref ref-type="bibr" rid="CR7">7</xref>], however, they didn’t thoroughly investigate the function and mechanism of <italic>HERV-E clone 4–1</italic> in SLE. In this study, we investigated the mechanism of <italic>HERV-E clone 4–1</italic> mRNA upregulation in CD4<sup>+</sup> T cells from SLE patients and its roles in SLE progression. First, we found <italic>NFAT1</italic> could induce <italic>HERV-E clone 4–1</italic> mRNA expression by binding to its 5′ LTR. <italic>NFAT1</italic>, which is a key factor of Ca<sup>2+</sup>/ calcineurin (CaN)/NFAT signaling pathways, was verified to be activated in SLE [<xref ref-type="bibr" rid="CR13">13</xref>]. We also demonstrated that <italic>NFAT1</italic> activity was upregulated in SLE and positively correlated with <italic>HERV-E clone 4–1</italic> mRNA expression. <italic>NFAT1</italic> are phosphorylated and reside in the cytoplasm in resting cells; upon stimulation, they are dephosphorylated by calcineurin, translocate to the nucleus, and become transcriptionally active [<xref ref-type="bibr" rid="CR29">29</xref>–<xref ref-type="bibr" rid="CR31">31</xref>]. Then the activated <italic>NFAT1</italic> can regulate transcription of some inflammatory cytokines such as IL-6, IL-8, TNF-α and interferon-γ (IFN-γ) [<xref ref-type="bibr" rid="CR32">32</xref>–<xref ref-type="bibr" rid="CR35">35</xref>]. Furthermore, we found TNF-α, IL-6, E2, LPS, UVB could upregulate <italic>NFAT1</italic> activity and <italic>HERV-E clone 4–1</italic> mRNA expression and these factors play critical roles in SLE [<xref ref-type="bibr" rid="CR14">14</xref>, <xref ref-type="bibr" rid="CR36">36</xref>–<xref ref-type="bibr" rid="CR38">38</xref>]. These results together may explain the roles of <italic>NFAT1</italic> in <italic>HERV-E clone 4–1</italic> mRNA expression in SLE.</p><p id="Par47">Adreno cortico hormones are an important class of anti-inflammatory/immunosuppressive drugs. They can inhibit the expression of TNF-α and IL-6 and decrease the activity of SLE [<xref ref-type="bibr" rid="CR39">39</xref>]. Ca<sup>2+</sup>/CaN/NFAT signaling is an important pathway in the T-cell activation of SLE and some calcineurin inhibitors such as cyclosporine A and tacrolimus have been used in the clinical treatment of SLE [<xref ref-type="bibr" rid="CR40">40</xref>]. Hydroxychloroquine, which could block Ca<sup>2+</sup>/CaN/NFAT signaling pathway through inhibiting the sustained Ca<sup>2+</sup> storage release from the endoplasmic reticulum [<xref ref-type="bibr" rid="CR41">41</xref>], was found to repress <italic>NFAT1</italic> activity and <italic>HERV-E clone 4–1</italic> expression. Prednisolone and hydroxychloroquine are first-line drugs in the treatment of SLE and all the patients followed-up got oral prednisolone and hydroxychloroquine treatment. These reasons interpret it well why <italic>HERV-E clone 4–1</italic> mRNA expressions decreased after prednisolone and hydroxychloroquine treatment. So, we hold that the upregulation of <italic>HERV-E clone 4–1</italic> mRNA is mainly due to the abnormal inflammation / immune status of SLE which involving many inflammatory cytokines and other risk factors. We also found that E2 could upregulate <italic>HERV-E clone 4–1</italic> mRNA expression via <italic>ER-α</italic>. <italic>ER-α</italic> is one of the estrogen receptors which can be activated by estrogen and regulate gene transcription in nucleus [<xref ref-type="bibr" rid="CR42">42</xref>]. Interestingly, HERV-E was upregulated in breast cancer and ovarian cancer [<xref ref-type="bibr" rid="CR15">15</xref>, <xref ref-type="bibr" rid="CR16">16</xref>] and this probably also has close relationship with E2 and <italic>ER-α</italic>. <italic>ER-α</italic> antagonist is also a good approach to restrain the expression of <italic>HERV-E clone 4–1</italic>. Taken together, we think these signaling pathways are good therapeutic targets for <italic>HERV-E clone 4–1</italic>.</p><p id="Par48">Some studies found the <italic>HERV-E clone 4–1</italic> 5’LTR was hypomethylated in CD4<sup>+</sup> T cells from SLE patients [<xref ref-type="bibr" rid="CR7">7</xref>–<xref ref-type="bibr" rid="CR9">9</xref>]. We found that DNA hypomethylation contributed to upregulation of <italic>HERV-E clone 4–1</italic> mRNA induced by <italic>NFAT1</italic> and <italic>ER-α</italic>. We think DNA hypomethylation of <italic>HERV-E clone 4–1</italic> 5’LTR is an indispensable factor that account for the upregulation of <italic>HERV-E clone 4–1</italic> mRNA for that upregulation of <italic>HERV-E clone 4–1</italic> mRNA mainly exists in SLE while not in some other diseases that involving <italic>NFAT1</italic> and <italic>ER-α</italic> activation.</p><p id="Par49">In this study, we found that <italic>HERV-E clone 4–1</italic> 3’LTR could act as natural miRNA sponges for <italic>miR-302d</italic> to restrain <italic>miR-302d</italic> activity. <italic>MiR-302d</italic> was proved to be downregulated in SLE patient monocytes and could inhibit the type I IFN pathway which was a major contributor to SLE pathogenesis via its target <italic>IRF-9</italic> [<xref ref-type="bibr" rid="CR21">21</xref>]. <italic>HERV-E clone 4–1</italic> 3’LTR could positively regulate <italic>MBD2</italic> expression by acting as a ceRNA of <italic>MBD2</italic> via <italic>miR-302d</italic> and <italic>HERV-E clone 4–1</italic> 3’LTR could induce DNA hypomethylation and IL-17 release via <italic>miR-302d</italic>/<italic>MBD2</italic> in CD4<sup>+</sup>T cells of SLE. DNA hypomethylation of immune cells in SLE is associated with immune dysfunction and play important roles in the initiation and development of SLE [<xref ref-type="bibr" rid="CR43">43</xref>, <xref ref-type="bibr" rid="CR44">44</xref>]. IL-17 is a proinflammatory cytokine produced by activated T cells and plays a crucial role in disease pathogenesis and represent an attractive therapeutic target for SLE [<xref ref-type="bibr" rid="CR27">27</xref>, <xref ref-type="bibr" rid="CR28">28</xref>]. Thus, we hold that <italic>HERV-E clone 4–1</italic> takes part in disease pathogenesis of SLE through <italic>miR-302d</italic>/<italic>MBD2</italic>/DNA hypomethylation and IL-17 signaling via its 3’LTR. So, <italic>HERV-E clone 4–1</italic> 3’LTR may be a potential therapeutic target of SLE. Taken together, we draw a network diagram hypothesis showing relationship between <italic>HERV-E clone 4–1</italic> and SLE which shows the important roles of <italic>HERV-E clone 4–1</italic> in SLE pathogenesis (Fig. <xref rid="Fig6" ref-type="fig">6</xref>).
<fig id="Fig6"><label>Fig. 6</label><caption><p>Network diagram hypothesis showing relationship between <italic>HERV-E clone 4–1</italic> and SLE</p></caption><graphic xlink:href="12964_2019_416_Fig6_HTML" id="MO6"></graphic></fig></p><p id="Par50">However, we should admit that we didn’t further investigate the role of <italic>HERV-E clone 4–1</italic> proteins and this is a shortcoming of this study. This mainly because there is no specific antibody for these proteins.</p></sec><sec id="Sec20"><title>Conclusions</title><p id="Par51">In conclusion, we found that <italic>HERV-E clone 4–1</italic> mRNA expression was upregulated in CD4<sup>+</sup> T cells from SLE patients and could act as a good biomarker for diagnosis of SLE. This is associated with the activation of Ca<sup>2+</sup>/CaN/<italic>NFAT1</italic> and E2/<italic>ER-α</italic> signaling pathway and DNA hypomethylation of <italic>HERV-E clone 4–1</italic> 5’LTR. <italic>HERV-E clone 4–1</italic> also takes part in disease pathogenesis of SLE through <italic>miR-302d</italic>/<italic>MBD2</italic>/DNA hypomethylation and IL-17 signaling via its 3’LTR. These signaling pathways may be potential therapeutic targets of SLE.</p></sec><sec sec-type="supplementary-material"><title>Additional file</title><sec id="Sec21"><p><supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="12964_2019_416_MOESM1_ESM.docx"><label>Additional file 1:</label><caption><p><bold>Figure S1</bold>. The structure of HERV-E clone 4–1. <bold>Table S1</bold>. Clinical characteristics of SLE patients and healthy controls. (DOCX 26 kb)</p></caption></media></supplementary-material></p></sec></sec></body><back><glossary><title>Abbreviations</title><def-list><def-item><term>5-aza C</term><def><p id="Par5">5-Azacytidine</p></def></def-item><def-item><term>AUC</term><def><p id="Par6">Area Under Curve</p></def></def-item><def-item><term>CaN</term><def><p id="Par7">Calcineurin</p></def></def-item><def-item><term>ChIP</term><def><p id="Par8">Chromatin immunoprecipitation</p></def></def-item><def-item><term>E2</term><def><p id="Par9">17β-estradiol/estradiol</p></def></def-item><def-item><term>ER-α</term><def><p id="Par10">Estrogen receptor-α</p></def></def-item><def-item><term>HCQ</term><def><p id="Par11">Hydroxychloroquine</p></def></def-item><def-item><term>HERV</term><def><p id="Par12">Human endogenous retroviruses</p></def></def-item><def-item><term>IFN-γ</term><def><p id="Par13">Interferon-γ</p></def></def-item><def-item><term>LPS</term><def><p id="Par14">Lipopolysaccharides</p></def></def-item><def-item><term>LTRs</term><def><p id="Par15">Long terminal repeats</p></def></def-item><def-item><term>MBD2</term><def><p id="Par16">Methyl-CpG binding domain protein 2</p></def></def-item><def-item><term>NFAT</term><def><p id="Par17">Nuclear factor of activated T cells</p></def></def-item><def-item><term>ORFs</term><def><p id="Par18">Open Reading Frames</p></def></def-item><def-item><term>PBMC</term><def><p id="Par19">Peripheral blood mononuclear cells</p></def></def-item><def-item><term>qRT-PCR</term><def><p id="Par20">Quantitative reverse transcription-PCR</p></def></def-item><def-item><term>ROC</term><def><p id="Par21">Operating Characteristic</p></def></def-item><def-item><term>SLE</term><def><p id="Par22">Systemic lupus erythematosus</p></def></def-item><def-item><term>SLEDAI</term><def><p id="Par23">SLE disease activity index</p></def></def-item><def-item><term>TNF-α</term><def><p id="Par24">Tumor necrosis factor-a</p></def></def-item><def-item><term>UVB</term><def><p id="Par25">Ultraviolet B</p></def></def-item></def-list></glossary><fn-group><fn><p><bold>Publisher’s Note</bold></p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p></fn><fn><p>Xin Wang, Chaoshuai Zhao and Chengzhong Zhang contributed equally to this work.</p></fn></fn-group><ack><title>Acknowledgements</title><p>Not applicable.</p></ack><notes notes-type="author-contribution"><title>Authors’ contributions</title><p>XW, CZ (Chaoshuai Zhao) and CZ (Chengzhong Zhang) designed and performed the experiments; ZW, XM, JS, YS and WS analyzed and interpreted the data; WS wrote the manuscript. ZW critically revised the manuscript. All authors read and approved the manuscript.</p></notes><notes notes-type="funding-information"><title>Funding</title><p>This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81573031 and 81773310).</p></notes><notes notes-type="data-availability"><title>Availability of data and materials</title><p>Not applicable.</p></notes><notes><title>Ethics approval and consent to participate</title><p id="Par52">This research was approved by the Institutional Research Ethics Committee of Shanghai General Hospital and abided by the ethical guidelines of the Declaration of Helsinki. 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