title: Demonstration of the presence of the "deleted" MIR122 gene in HepG2 cells.
abstract: 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.
authors: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, UCL, London, WC1E 6JF, United Kingdom; Medical Biochemistry Department, Alazhar University, Damietta, 34511, Egypt.
authors: Ibrahim A Y
authors: IA
authors: Hamad
authors: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, UCL, London, WC1E 6JF, United Kingdom.
authors: Yue
authors: Y
authors: Fei
authors: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, UCL, London, WC1E 6JF, United Kingdom.
authors: Anastasia Z
authors: AZ
authors: Kalea
authors: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, UCL, London, WC1E 6JF, United Kingdom; Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology (HUST), Wuhan, 430074, PR China.
authors: Dan
authors: D
authors: Yin
authors: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, UCL, London, WC1E 6JF, United Kingdom.
authors: Andrew J P
authors: AJ
authors: Smith
authors: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, UCL, London, WC1E 6JF, United Kingdom.
authors: Jutta
authors: J
authors: Palmen
authors: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, UCL, London, WC1E 6JF, United Kingdom.
authors: Steve E
authors: SE
authors: Humphries
authors: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, UCL, London, WC1E 6JF, United Kingdom.
authors: Philippa J
authors: PJ
authors: Talmud
authors: Centre for Cardiovascular Genetics, BHF Laboratories, Institute of Cardiovascular Science, UCL, London, WC1E 6JF, United Kingdom.
authors: Ann P
authors: AP
authors: Walker
mesh_terms: D003001:Cloning, Molecular; D017353:Gene Deletion; D018628:Gene Dosage; D006239:Haplotypes; D056945:Hep G2 Cells; D006579:Heterozygote; D006801:Humans; D055029:Inverted Repeat Sequences; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic
chemical_list: C531907:MIRN122 microRNA, human; D035683:MicroRNAs