Demonstration of the Presence of the “Deleted” <i>MIR122</i> Gene in HepG2 Cells

<div><p>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.</p></div

The “deleted” <i>MIR122</i> gene is present in two copies in HepG2 cells.

<p>A schematic diagram illustrates the human <i>MIR122</i> genomic locus. Li et al (2011) identified the pri-mir-122 promoter and the two DR-1 sites which were investigated by FAIRE in this study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122471#pone.0122471.ref014" target="_blank">14</a>]. The position of the pre-mir-122 stem-loop region, previously reported to be deleted in HepG2 cells, is indicated. FAIRE showed that Huh-7 cells had 6.2 ± 0.19- and 2.7 ± 0.01- fold more accessible chromatin at the DR1-1 (proximal) and DR1-2 (distal) sites than HepG2 (p = 0.03 and 0.001), respectively. The ENCODE DNase I hypersensitivity (DNAse HS, relative units; Duke University) annotations of <i>MIR122</i> are shown as density signals for Huh-7 and HepG2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122471#pone.0122471.ref022" target="_blank">22</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122471#pone.0122471.ref023" target="_blank">23</a>]. Consistent with the FAIRE results, these annotations showed a DNase I hypersensitivity site in the pri-mir-122 promoter in Huh-7 that was not seen in HepG2. The region was sequenced in 13 reactions (overlapping horizontal bars) and shown to be present in both Huh-7 and HepG2 cell lines (HepG2 isolates 1, 2 and 3). Ten SNPs spanning the region were heterozygous indicating that DNA was present in two copies. The positions of the miR-122 gene (upstream) and miR-122 gene (downstream) primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122471#pone.0122471.t001" target="_blank">Table 1</a>) previously used to report the HepG2 <i>MIR122</i> “deletion” [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122471#pone.0122471.ref009" target="_blank">9</a>] are indicated by arrowheads; the downstream primer was located within an AluJb repeat sequence. The widely-reported deficiency of miR-122 expression in HepG2 cells is not caused by a <i>MIR122</i> deletion, but may be related to a less accessible chromatin conformation in HepG2 than Huh-7 cells. Scale bar, 2kb.</p

Cloned pre-mir-122 stem-loop region sequences from HepG2 DNA show two different haplotypes.

<p>(A) Cloned DNA sequences obtained after amplification with Taq polymerase. Two haplotypes (differently shaded) were observed for HepG2, consistent with the presence of two alleles across this region. However, among the eight HepG2 and Huh-7 clones, six sequence differences to the reference genome assembly were detected (*), so cloning was repeated using a proofreading DNA polymerase. (B) Cloned DNA sequences obtained after amplification with Phusion high fidelity DNA polymerase. Essentially the same two haplotypes of HepG2 were seen, but three novel single nucleotide substitution variants were detected and in a fourth clone, the rs9966765 allele did not correspond to the background haplotype observed. The reported error rate of Phusion High-Fidelity DNA Polymerase (GC Buffer) is 9.5 x 10^-7 errors / base pair / PCR cycle (New England Biolabs). SNPs rs9966765 and rs1135519 are located upstream of the pre-mir-122 stem-loop region; their respective alleles are shown. The genomic positions on chromosome 18 (GRCh37/hg19 (Feb. 2009) human genome assembly) of non-SNP sequence variants and the alleles observed are shown; (T)_n refers to the length (base pairs) of the polymorphic poly(T) tract. *, position showing a sequence variant not corresponding to the predominant haplotypes observed.</p

Relative Expression of <i>PLA2G2A</i> mRNA in tissues from the ASAP study.

<p>This chart shows the relative expression of <i>PLA2G2A</i> mRNA across 5 tissues; liver, aorta intima-media, aorta adventitia, mammary artery intima-media and heart, in 223 patients undergoing aortic valve surgery in the Advanced Study of Aortic Pathology (ASAP) study.</p

TaqMan Gene Expression Analysis for rs11573156 C and G.

<p>TaqMan gene expression assay results for rs11573156 C vs. rs11573156 G expression levels across <i>PLA2G2A</i> exon 1–2 and exon 5–6. The X axis scale represents ΔCt, the number of amplification cycles required to reach a pre-determined threshold of fluorescent signal. All samples were normalised to the housekeeping gene <i>GAPDH</i>.</p

Luciferase Assay Activity Analysis Comparing rs3767221 T >G.

<p>The difference in luciferase activity (Relative Light Units) between the T and G alleles of rs3767221 (forward orientation), normalised to pGL3 expression vector. Rs3767221 G (rare) shows a 55% lower Luc activity compared to the T allele (wild type) and is compared to the promoter-free vector pGL3-Basic for baseline reference.</p

Luciferase Assay Activity Analysis Comparing rs11573156 C >G.

<p>The difference in luciferase activity (Relative Light Units) between the C and G alleles of rs11573156 normalised to pGL3-Basic. There is no pre-existing promoter in this vector and both inserted constructs for C and G variants give very low levels of luciferase when compared with pGL3-Basic for baseline reference.</p

Figure 3

<p><b>a. Differential </b><b><i>PLA2G2A</i></b><b> expression of rs10732279 in the Liver.</b> Results from the Advanced Study of Aortic Pathology of the overall differential liver expression of <i>PLA2G2A</i> according to rs10732279 (A>G) genotype. Rare G carriers have significantly higher <i>PLA2G2A</i> expression than common A homozygotes (p = 1.67×10⁻¹⁷). <b>b. </b><b>Predicted Differential </b><b><i>PLA2G2A</i></b><b> expression of rs3767221 in the Liver.</b> Results from the Advanced Study of Aortic Pathology relative to the 1000 Genomes Project imputed SNPs for <i>PLA2G2A</i>. The figure shows the overall differential expression predicted for rs3767221 (T>G) in the liver. Common T carriers have significantly higher <i>PLA2G2A</i> expression than rare G homozygotes (p = 3.6×10⁻⁴).</p

EMSA Analysis of Potential Differential Binding for rs11573156 C and G Variants.

<p>EMSA image of rs11573156 C and G alleles showing no transcription factor binding. Lane 1 is SREBP 1 positive control that shows specific bands that are competed out with an unlabelled SREBP 1 consensus sequence in lane 2. Lanes 3 and 4 show rs11573156 C (wild type) labelled and competed with unlabelled C competitor respectively. There is no specific binding. Lanes 5 and 6 represent rs11573156 G (rare) labelled and competed with unlabelled G competitor respectively, again with no specific binding. Lane 7 represents a second positive control with Sp1 specific binding and in Lane 8, labelled Sp1 with unlabelled Sp1 competitor that competes out Sp1 specific bands.</p

a. ASAP <i>PLA2G2A</i> Expression Analysis for Different rs10732279 Genotypes.

<p>Results from the ASAP study; liver expression levels of exon-specific <i>PLA2G2A</i> probe sets stratified by rs10732279 genotype. <i>PLA2G2A</i> is covered by 9 exon-specific probe sets (1 in exon 2, and two each in exon 3–6) <b>b.. </b><b>The Known Protein Coding Transcripts for </b><b><i>PLA2G2A.</i></b> Known transcribed <i>PLA2G2A</i> transcripts showing alternate exon 2 skipping from the UCSC Genome Browser (UCSC Genome Browser). <b>c.. </b><b><i>PLA2G2A</i></b><b> Exon-Specific Expression in the Liver.</b> Results from the ASAP study; Liver expression association between exon-specific <i>PLA2G2A</i> probe sets according to rs10732279 genotype. The y-axis give the –log10 (p-value) for the <i>PLA2G2A</i> association for rs10732279, using an additive model. These values relate to the exon specific absolute expression levels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041139#pone-0041139-g009" target="_blank">Figure 9a</a>.</p