Demonstration of the presence of the "deleted" MIR122 gene in HepG2 cells
MicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionari...
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| description | MicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells. |
| doi_str_mv | 10.1371/journal.pone.0122471 |
| format | Article |
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It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0122471</identifier><identifier>PMID: 25811611</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Accessibility ; Analysis ; Annotations ; Bioinformatics ; Biological activity ; Biotechnology ; Cell culture ; Cell growth ; Cell lines ; Cholesterol ; Chromatin ; Clonal deletion ; Cloning ; Cloning, Molecular ; Deoxyribonuclease ; Deoxyribonucleic acid ; DNA ; Gene Deletion ; Gene Dosage ; Gene expression ; Genes ; Genetics ; Genomes ; Genomics ; Genotypes ; Haplotypes ; Hep G2 Cells ; Hepatitis ; Hepatitis C ; Hepatitis C virus ; Hepatocytes ; Hepatology ; Heterozygote ; Humans ; Inverted Repeat Sequences ; Iron ; Laboratories ; Lipid metabolism ; Liver ; Liver cancer ; Metabolism ; MicroRNAs ; MicroRNAs - chemistry ; MicroRNAs - genetics ; miRNA ; Nucleotide sequence ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Public health ; Ribonucleic acid ; RNA ; RNA polymerase ; Science ; Single nucleotide polymorphisms ; Single-nucleotide polymorphism ; Viruses</subject><ispartof>PloS one, 2015-03, Vol.10 (3), p.e0122471</ispartof><rights>COPYRIGHT 2015 Public Library of Science</rights><rights>2015 Hamad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2015 Hamad et al 2015 Hamad et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-45677732dbe33ad1f4fc716fbe077d51794a84e8fa450804e0ba2ed346a16213</citedby><cites>FETCH-LOGICAL-c692t-45677732dbe33ad1f4fc716fbe077d51794a84e8fa450804e0ba2ed346a16213</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374784/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374784/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23847,27903,27904,53769,53771,79346,79347</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25811611$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Pfeffer, Sebastien</contributor><creatorcontrib>Hamad, Ibrahim A Y</creatorcontrib><creatorcontrib>Fei, Yue</creatorcontrib><creatorcontrib>Kalea, Anastasia Z</creatorcontrib><creatorcontrib>Yin, Dan</creatorcontrib><creatorcontrib>Smith, Andrew J P</creatorcontrib><creatorcontrib>Palmen, Jutta</creatorcontrib><creatorcontrib>Humphries, Steve E</creatorcontrib><creatorcontrib>Talmud, Philippa J</creatorcontrib><creatorcontrib>Walker, Ann P</creatorcontrib><title>Demonstration of the presence of the "deleted" MIR122 gene in HepG2 cells</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>MicroRNA 122 (miR-122) is highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells.</description><subject>Accessibility</subject><subject>Analysis</subject><subject>Annotations</subject><subject>Bioinformatics</subject><subject>Biological activity</subject><subject>Biotechnology</subject><subject>Cell culture</subject><subject>Cell growth</subject><subject>Cell lines</subject><subject>Cholesterol</subject><subject>Chromatin</subject><subject>Clonal deletion</subject><subject>Cloning</subject><subject>Cloning, Molecular</subject><subject>Deoxyribonuclease</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>Gene Deletion</subject><subject>Gene Dosage</subject><subject>Gene expression</subject><subject>Genes</subject><subject>Genetics</subject><subject>Genomes</subject><subject>Genomics</subject><subject>Genotypes</subject><subject>Haplotypes</subject><subject>Hep G2 Cells</subject><subject>Hepatitis</subject><subject>Hepatitis C</subject><subject>Hepatitis C virus</subject><subject>Hepatocytes</subject><subject>Hepatology</subject><subject>Heterozygote</subject><subject>Humans</subject><subject>Inverted Repeat Sequences</subject><subject>Iron</subject><subject>Laboratories</subject><subject>Lipid metabolism</subject><subject>Liver</subject><subject>Liver cancer</subject><subject>Metabolism</subject><subject>MicroRNAs</subject><subject>MicroRNAs - chemistry</subject><subject>MicroRNAs - genetics</subject><subject>miRNA</subject><subject>Nucleotide sequence</subject><subject>Polymorphism, Single Nucleotide</subject><subject>Promoter Regions, Genetic</subject><subject>Public health</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA polymerase</subject><subject>Science</subject><subject>Single nucleotide polymorphisms</subject><subject>Single-nucleotide 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of the presence of the "deleted" MIR122 gene in HepG2 cells</title><author>Hamad, Ibrahim A Y ; Fei, Yue ; Kalea, Anastasia Z ; Yin, Dan ; Smith, Andrew J P ; Palmen, Jutta ; Humphries, Steve E ; Talmud, Philippa J ; Walker, Ann P</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-45677732dbe33ad1f4fc716fbe077d51794a84e8fa450804e0ba2ed346a16213</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>Accessibility</topic><topic>Analysis</topic><topic>Annotations</topic><topic>Bioinformatics</topic><topic>Biological activity</topic><topic>Biotechnology</topic><topic>Cell culture</topic><topic>Cell growth</topic><topic>Cell lines</topic><topic>Cholesterol</topic><topic>Chromatin</topic><topic>Clonal deletion</topic><topic>Cloning</topic><topic>Cloning, Molecular</topic><topic>Deoxyribonuclease</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>Gene 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highly expressed in the liver where it influences diverse biological processes and pathways, including hepatitis C virus replication and metabolism of iron and cholesterol. It is processed from a long non-coding primary transcript (~7.5 kb) and the gene has two evolutionarily-conserved regions containing the pri-mir-122 promoter and pre-mir-122 hairpin region. Several groups reported that the widely-used hepatocytic cell line HepG2 had deficient expression of miR-122, previously ascribed to deletion of the pre-mir-122 stem-loop region. We aimed to characterise this deletion by direct sequencing of 6078 bp containing the pri-mir-122 promoter and pre-mir-122 stem-loop region in HepG2 and Huh-7, a control hepatocytic cell line reported to express miR-122, supported by sequence analysis of cloned genomic DNA. In contrast to previous findings, the entire sequence was present in both cell lines. Ten SNPs were heterozygous in HepG2 indicating that DNA was present in two copies. Three validation isolates of HepG2 were sequenced, showing identical genotype to the original in two, whereas the third was different. Investigation of promoter chromatin status by FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the proximal (HNF4α-binding) and distal DR1 transcription factor sites, compared to HepG2 cells (p=0.03 and 0.001, respectively). This was substantiated by ENCODE genome annotations, which showed a DNAse I hypersensitive site in the pri-mir-122 promoter in Huh-7 that was absent in HepG2 cells. While the origin of the reported deletion is unclear, cell lines should be obtained from a reputable source and used at low passage number to avoid discrepant results. Deficiency of miR-122 expression in HepG2 cells may be related to a relative deficiency of accessible promoter chromatin in HepG2 versus Huh-7 cells.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>25811611</pmid><doi>10.1371/journal.pone.0122471</doi><oa>free_for_read</oa></addata></record> |
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| ispartof | PloS one, 2015-03, Vol.10 (3), p.e0122471 |
| issn | 1932-6203 1932-6203 |
| language | eng |
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| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| subjects | Accessibility Analysis Annotations Bioinformatics Biological activity Biotechnology Cell culture Cell growth Cell lines Cholesterol Chromatin Clonal deletion Cloning Cloning, Molecular Deoxyribonuclease Deoxyribonucleic acid DNA Gene Deletion Gene Dosage Gene expression Genes Genetics Genomes Genomics Genotypes Haplotypes Hep G2 Cells Hepatitis Hepatitis C Hepatitis C virus Hepatocytes Hepatology Heterozygote Humans Inverted Repeat Sequences Iron Laboratories Lipid metabolism Liver Liver cancer Metabolism MicroRNAs MicroRNAs - chemistry MicroRNAs - genetics miRNA Nucleotide sequence Polymorphism, Single Nucleotide Promoter Regions, Genetic Public health Ribonucleic acid RNA RNA polymerase Science Single nucleotide polymorphisms Single-nucleotide polymorphism Viruses |
| title | Demonstration of the presence of the "deleted" MIR122 gene in HepG2 cells |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-28T03%3A58%3A23IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Demonstration%20of%20the%20presence%20of%20the%20%22deleted%22%20MIR122%20gene%20in%20HepG2%20cells&rft.jtitle=PloS%20one&rft.au=Hamad,%20Ibrahim%20A%20Y&rft.date=2015-03-26&rft.volume=10&rft.issue=3&rft.spage=e0122471&rft.pages=e0122471-&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0122471&rft_dat=%3Cgale_plos_%3EA421818162%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1667003746&rft_id=info:pmid/25811611&rft_galeid=A421818162&rft_doaj_id=oai_doaj_org_article_e5b8623e62104b3c870bf244d6f8df0a&rfr_iscdi=true |