{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,2]],"date-time":"2026-11-02T12:17:25Z","timestamp":1793621845634,"version":"4.5.0"},"reference-count":49,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2024,6,6]],"date-time":"2024-06-06T00:00:00Z","timestamp":1717632000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"NHLBI Division of Intramural Research funds","doi-asserted-by":"publisher","award":["1ZIAHL006103"],"award-info":[{"award-number":["1ZIAHL006103"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"NHLBI Division of Intramural Research funds","doi-asserted-by":"publisher","award":["1ZIAHL006159"],"award-info":[{"award-number":["1ZIAHL006159"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["ncRNA"],"abstract":"<jats:p>Fibrotic liver features excessive deposition of extracellular matrix (ECM), primarily produced from \u201cactivated\u201d hepatic stellate cells (HSCs). While targeting human HSCs (hHSCs) in fibrosis therapeutics shows promise, the overall understanding of hHSC activation remains limited, in part because it is very challenging to define the role of human long non-coding RNAs (lncRNAs) in hHSC activation. To address this challenge, we identified another cell type that acts via a diverse gene network to promote fibrogenesis. Then, we identified the lncRNAs that were differentially regulated in activated hHSCs and the other profibrotic cell. Next, we conducted concurrent analysis to identify those lncRNAs that were specifically involved in fibrogenesis. We tested and confirmed that transdifferentiation of vascular smooth muscle cells (VSMCs) represents such a process. By overlapping TGF\u03b2-regulated lncRNAs in multiple sets of hHSCs and VSMCs, we identified a highly selected list of lncRNA candidates that could specifically play a role in hHSC activation. We experimentally characterized one human lncRNA, named CARMN, which was significantly regulated by TGF\u03b2 in all conditions above. CARMN knockdown significantly reduced the expression levels of a panel of marker genes for hHSC activation, as well as the levels of ECM deposition and hHSC migration. Conversely, gain of function of CARMN using CRISPR activation (CRISPR-a) yielded the completely opposite effects. Taken together, our work addresses a bottleneck in identifying human lncRNAs that specifically play a role in hHSC activation and provides a framework to effectively select human lncRNAs with significant pathophysiological role.<\/jats:p>","DOI":"10.3390\/ncrna10030034","type":"journal-article","created":{"date-parts":[[2024,6,6]],"date-time":"2024-06-06T06:40:26Z","timestamp":1717656026000},"page":"34","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["An Integrative Transcriptome Subtraction Strategy to Identify Human lncRNAs That Specifically Play a Role in Activation of Human Hepatic Stellate Cells"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7587-7921","authenticated-orcid":false,"given":"Yonghe","family":"Ma","sequence":"first","affiliation":[{"name":"Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2136-1898","authenticated-orcid":false,"given":"Jamie","family":"Harris","sequence":"additional","affiliation":[{"name":"Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Li","sequence":"additional","affiliation":[{"name":"Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chengfei","family":"Jiang","sequence":"additional","affiliation":[{"name":"Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hang","family":"Sun","sequence":"additional","affiliation":[{"name":"Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haiming","family":"Cao","sequence":"additional","affiliation":[{"name":"Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,6,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2212","DOI":"10.1016\/S0140-6736(20)32511-3","article-title":"Non-alcoholic fatty liver disease","volume":"397","author":"Powell","year":"2021","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1159\/000282080","article-title":"Epidemiology of non-alcoholic fatty liver disease","volume":"28","author":"Bellentani","year":"2010","journal-title":"Dig. Dis."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1769","DOI":"10.1053\/j.gastro.2016.02.066","article-title":"Pathogenesis of Nonalcoholic Steatohepatitis","volume":"150","author":"Machado","year":"2016","journal-title":"Gastroenterology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"908","DOI":"10.1038\/s41591-018-0104-9","article-title":"Mechanisms of NAFLD development and therapeutic strategies","volume":"24","author":"Friedman","year":"2018","journal-title":"Nat. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1016\/j.jhep.2023.03.038","article-title":"An integrated view of anti-inflammatory and antifibrotic targets for the treatment of NASH","volume":"79","author":"Tacke","year":"2023","journal-title":"J. Hepatol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1111\/cts.12839","article-title":"Evolving Role for Pharmacotherapy in NAFLD\/NASH","volume":"14","author":"Attia","year":"2021","journal-title":"Clin. Transl. Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"C630","DOI":"10.1152\/ajpcell.00123.2022","article-title":"Role of hepatic lipid species in the progression of nonalcoholic fatty liver disease","volume":"323","author":"Deng","year":"2022","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1016\/0003-2697(87)90673-7","article-title":"Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan","volume":"161","author":"Friedman","year":"1987","journal-title":"Anal. Biochem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1007\/BF01324802","article-title":"The hepatic stellate (Ito) cell: Its role in human liver disease","volume":"430","author":"Hautekeete","year":"1997","journal-title":"Virchows Arch."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1034\/j.1600-0676.2002.01726.x","article-title":"Mesenchymal cells in the liver--one cell type or two?","volume":"22","author":"Ramadori","year":"2002","journal-title":"Liver"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1016\/j.jhep.2010.02.005","article-title":"Fibrogenic signalling in hepatic stellate cells","volume":"52","author":"Mann","year":"2010","journal-title":"J. Hepatol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1104","DOI":"10.1016\/j.jhep.2017.06.001","article-title":"Hepatic stellate cell activation and pro-fibrogenic signals","volume":"67","author":"Gandhi","year":"2017","journal-title":"J. Hepatol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1038\/nrgastro.2017.38","article-title":"Mechanisms of hepatic stellate cell activation","volume":"14","author":"Tsuchida","year":"2017","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Zisser, A., Ipsen, D.H., and Tveden-Nyborg, P. (2021). Hepatic Stellate Cell Activation and Inactivation in NASH-Fibrosis-Roles as Putative Treatment Targets?. Biomedicines, 9.","DOI":"10.3390\/biomedicines9040365"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1038\/s41597-020-00659-z","article-title":"lncRNAKB, a knowledgebase of tissue-specific functional annotation and trait association of long noncoding RNA","volume":"7","author":"Seifuddin","year":"2020","journal-title":"Sci. Data"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1038\/ng.3192","article-title":"The landscape of long noncoding RNAs in the human transcriptome","volume":"47","author":"Iyer","year":"2015","journal-title":"Nat. Genet."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1038\/s41580-020-00315-9","article-title":"Gene regulation by long non-coding RNAs and its biological functions","volume":"22","author":"Statello","year":"2021","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"744","DOI":"10.1002\/hep.27043","article-title":"Long noncoding RNA in liver diseases","volume":"60","author":"Takahashi","year":"2014","journal-title":"Hepatology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1038\/nature17674","article-title":"Feedback modulation of cholesterol metabolism by the lipid-responsive non-coding RNA LeXis","volume":"534","author":"Sallam","year":"2016","journal-title":"Nature"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1298","DOI":"10.1016\/j.cell.2013.02.012","article-title":"Long noncoding RNAs: Cellular address codes in development and disease","volume":"152","author":"Batista","year":"2013","journal-title":"Cell"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/s41467-019-13688-z","article-title":"In vivo functional analysis of non-conserved human lncRNAs associated with cardiometabolic traits","volume":"11","author":"Ruan","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e136336","DOI":"10.1172\/JCI136336","article-title":"Identification of human long noncoding RNAs associated with nonalcoholic fatty liver disease and metabolic homeostasis","volume":"131","author":"Ruan","year":"2021","journal-title":"J. Clin. Investig."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1038\/nature12943","article-title":"The evolution of lncRNA repertoires and expression patterns in tetrapods","volume":"505","author":"Necsulea","year":"2014","journal-title":"Nature"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.tig.2014.01.004","article-title":"The four dimensions of noncoding RNA conservation","volume":"30","author":"Diederichs","year":"2014","journal-title":"Trends Genet."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1247\/csf.28.105","article-title":"Hepatic stellate cells: Unique characteristics in cell biology and phenotype","volume":"28","author":"Sato","year":"2003","journal-title":"Cell Struct. Funct."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/978-1-0716-3207-9_1","article-title":"Isolation, Purification, and Culture of Primary Murine Hepatic Stellate Cells: An Update","volume":"2669","author":"Meurer","year":"2023","journal-title":"Methods Mol. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1007\/978-1-4939-7113-8_11","article-title":"Isolation and Culture of Primary Murine Hepatic Stellate Cells","volume":"1627","author":"Weiskirchen","year":"2017","journal-title":"Methods Mol. Biol."},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Dang, T.M., Le, V.T., Do, H.Q., Nguyen, V.T., Holterman, A.X.L., Dang, L.T.T., Phan, N.C.L., Pham, P.V., Hoang, S.N., and Le, L.T. (2021). Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse. Biosci. Rep., 41.","DOI":"10.1042\/BSR20202514"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"d793","DOI":"10.2741\/gressner","article-title":"Roles of TGF-beta in hepatic fibrosis","volume":"7","author":"Gressner","year":"2002","journal-title":"Front. Biosci."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2209","DOI":"10.1016\/j.ajpath.2019.07.019","article-title":"Smooth Muscle alpha-Actin Deficiency Leads to Decreased Liver Fibrosis via Impaired Cytoskeletal Signaling in Hepatic Stellate Cells","volume":"189","author":"Rockey","year":"2019","journal-title":"Am. J. Pathol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1599","DOI":"10.1096\/fj.14-268425","article-title":"Lysyl oxidase activity contributes to collagen stabilization during liver fibrosis progression and limits spontaneous fibrosis reversal in mice","volume":"30","author":"Liu","year":"2016","journal-title":"FASEB J."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5919","DOI":"10.1074\/jbc.274.9.5919","article-title":"Alternatively spliced EDA segment regulates fibronectin-dependent cell cycle progression and mitogenic signal transduction","volume":"274","author":"Manabe","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1016\/j.ebiom.2019.04.036","article-title":"A PDGFRbeta-based score predicts significant liver fibrosis in patients with chronic alcohol abuse, NAFLD and viral liver disease","volume":"43","author":"Lambrecht","year":"2019","journal-title":"EBioMedicine"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"615","DOI":"10.47162\/RJME.61.3.01","article-title":"Hepatic stellate cells\u2014From past till present: Morphology, human markers, human cell lines, behavior in normal and liver pathology","volume":"61","author":"Sufletel","year":"2020","journal-title":"Rom. J. Morphol. Embryol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1016\/S0168-8278(02)00103-4","article-title":"Reversal of activation of human myofibroblast-like cells by culture on a basement membrane-like substrate","volume":"37","author":"Sohara","year":"2002","journal-title":"J. Hepatol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"692","DOI":"10.1161\/CIRCRESAHA.115.306361","article-title":"Vascular Smooth Muscle Cells in Atherosclerosis","volume":"118","author":"Bennett","year":"2016","journal-title":"Circ. Res."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"812","DOI":"10.1161\/ATVBAHA.107.159327","article-title":"Role of smooth muscle cells in the initiation and early progression of atherosclerosis","volume":"28","author":"Doran","year":"2008","journal-title":"Arterioscler. Thromb. Vasc. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"912","DOI":"10.1038\/s42255-019-0102-3","article-title":"Endothelial TGF-beta signalling drives vascular inflammation and atherosclerosis","volume":"1","author":"Chen","year":"2019","journal-title":"Nat. Metab."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.cellsig.2018.09.004","article-title":"TGFbeta, smooth muscle cells and coronary artery disease: A review","volume":"53","author":"Low","year":"2019","journal-title":"Cell Signal"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e1","DOI":"10.1161\/ATVBAHA.111.238410","article-title":"Transforming growth factor-beta signaling in myogenic cells regulates vascular morphogenesis, differentiation, and matrix synthesis","volume":"32","author":"Jaffe","year":"2012","journal-title":"Arterioscler. Thromb. Vasc. Biol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.jhep.2003.12.006","article-title":"On the origin of stellate cells: Mesodermal, endodermal or neuro-ectodermal?","volume":"40","author":"Geerts","year":"2004","journal-title":"J. Hepatol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"784","DOI":"10.1111\/j.1432-0436.2007.00260.x","article-title":"Hepatic stellate cells display a functional vascular smooth muscle cell phenotype in a three-dimensional co-culture model with endothelial cells","volume":"76","author":"Wirz","year":"2008","journal-title":"Differentiation"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"110597","DOI":"10.1016\/j.ygeno.2023.110597","article-title":"SNAI1-activated long non-coding RNA LINC01711 promotes hepatic fibrosis cell proliferation and migration by regulating XYLT1","volume":"115","author":"Bao","year":"2023","journal-title":"Genomics"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Ilieva, M., Miller, H.E., Agarwal, A., Paulus, G.K., Madsen, J.H., Bishop, A.J.R., Kauppinen, S., and Uchida, S. (2022). FibroDB: Expression Analysis of Protein-Coding and Long Non-Coding RNA Genes in Fibrosis. Noncoding RNA, 8.","DOI":"10.3390\/ncrna8010013"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.yjmcc.2015.09.016","article-title":"CARMEN, a human super enhancer-associated long noncoding RNA controlling cardiac specification, differentiation and homeostasis","volume":"89","author":"Ounzain","year":"2015","journal-title":"J. Mol. Cell Cardiol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1856","DOI":"10.1161\/CIRCULATIONAHA.121.055949","article-title":"CARMN Is an Evolutionarily Conserved Smooth Muscle Cell-Specific LncRNA That Maintains Contractile Phenotype by Binding Myocardin","volume":"144","author":"Dong","year":"2021","journal-title":"Circulation"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2399","DOI":"10.1161\/ATVBAHA.120.315911","article-title":"A Smooth Muscle Cell-Enriched Long Noncoding RNA Regulates Cell Plasticity and Atherosclerosis by Interacting with Serum Response Factor","volume":"41","author":"Ni","year":"2021","journal-title":"Arterioscler. Thromb. Vasc. Biol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1007\/978-1-4939-2809-5_18","article-title":"Analysis of Cell Proliferation and Homeostasis Using EdU Labeling","volume":"1323","author":"Flomerfelt","year":"2016","journal-title":"Methods Mol. Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"468","DOI":"10.1126\/science.aae0047","article-title":"Xist recruits the X chromosome to the nuclear lamina to enable chromosome-wide silencing","volume":"354","author":"Chen","year":"2016","journal-title":"Science"}],"container-title":["Non-Coding RNA"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2311-553X\/10\/3\/34\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T14:54:42Z","timestamp":1760108082000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2311-553X\/10\/3\/34"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,6,6]]},"references-count":49,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2024,6]]}},"alternative-id":["ncrna10030034"],"URL":"https:\/\/doi.org\/10.3390\/ncrna10030034","relation":{},"ISSN":["2311-553X"],"issn-type":[{"value":"2311-553X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,6,6]]}}}