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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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Autores principales: 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.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374784/
https://www.ncbi.nlm.nih.gov/pubmed/25811611
http://dx.doi.org/10.1371/journal.pone.0122471
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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_facet 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 Hamad, Ibrahim A. Y.
collection PubMed
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.
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spelling pubmed-43747842015-04-04 Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells 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. PLoS One Research Article 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. Public Library of Science 2015-03-26 /pmc/articles/PMC4374784/ /pubmed/25811611 http://dx.doi.org/10.1371/journal.pone.0122471 Text en © 2015 Hamad et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
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.
Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
title Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
title_full Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
title_fullStr Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
title_full_unstemmed Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
title_short Demonstration of the Presence of the “Deleted” MIR122 Gene in HepG2 Cells
title_sort demonstration of the presence of the “deleted” mir122 gene in hepg2 cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374784/
https://www.ncbi.nlm.nih.gov/pubmed/25811611
http://dx.doi.org/10.1371/journal.pone.0122471
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