MicroRNA-224 is up-regulated in hepatocellular carcinoma through epigenetic mechanisms - PubMed (original) (raw)
MicroRNA-224 is up-regulated in hepatocellular carcinoma through epigenetic mechanisms
Yu Wang et al. FASEB J. 2012 Jul.
Abstract
MicroRNA-224 (miR-224) is one of the most commonly up-regulated microRNAs in hepatocellular carcinoma (HCC), which affects crucial cellular processes such as apoptosis and cell proliferation. In this study, we aim to elucidate the molecular mechanism that leads to the overexpression of miR-224 in HCC. We examined the transcript expression of miR-224 and neighboring miR-452 and genes on chromosome Xq28 in tumor and paired adjacent nontumorous tissues from 100 patients with HCC and found that miR-224 is coordinately up-regulated with its neighboring microRNA (miRNA) and genes. This coordinated up-regulation of miRNAs and genes at the Xq28 locus can be mimicked in nontransformed immortalized human liver cells by the introduction of histone deacetylase (HDAC) inhibitors, which resulted in a corresponding increase in histone H3 acetylation in this region. This miR-224-residing locus in Xq28 is reciprocally regulated by HDAC1, HDAC3, and histone acetylase protein, E1A binding protein p300 (EP300). Notably, in HCC tumors that significantly overexpress microRNA-224, EP300 is also overexpressed and displays increased binding to the Xq28 locus. In transformed HCC cells, high miR-224 expression can be attenuated through the inhibition of EP300, using either siRNA or the specific drug C646. In summary, overexpression of EP300 may account, in part, for the up-regulation of miR-224 expression in patients with HCC.
Figures
Figure 1.
miR-224 is coordinately up-regulated with neighboring miRNAs and genes on Xq28 in hepatocellular carcinoma patients. A) Genomic structure of 200-kb region of Xq28 consisting of miR-224/miR-452 cluster residing in intron 6 of GABRE gene, flanked by two cancer and testis antigens, MAGEA4 and MAGEA5. B) Relative expression of miR-224 associated miRNAs and genes on Xq28 and unrelated miR-16, miR-100 and U6, in 40 normal liver samples (open box), 100 paired adjacent nontumor samples (light shaded box), and 100 HCC tumor samples (dark shaded box), measured using RT-qPCR and normalized against β-actin (for genes) or RNU48 (for miRNAs) as endogenous control. Data were normalized against the median expression of each miRNA or gene in normal liver samples and presented as box-and-whisker plots with the box spanning the 25th to 75th percentile and the solid line representing the median. Outliers were removed from the plots. *P < 0.05, **P < 0.01, ***P < 0.001. C) Scatterplots showing the representative correlation of relative fold change of transcript expression (in log2 scale) between miR-224 and miR-452 (I) and miR-224 and GABRE (II), in tumor vs. paired adjacent nontumor tissues from 100 patients with HCC. Each spot represents data from one patient with HCC; solid line is the linear regression line. See Table 2 for pairwise Pearson correlation of the relative fold change of Xq28 genes/miRNAs between tumor vs. paired adjacent nontumorous tissues. D) Box-and-whisker plots showing the relative genomic DNA content of Xq28 measured with qPCR using specific primers targeting promoters of MAGEA4, MAGEA5, and GABRE and the miR-224- and mir-452-residing GABRE intron 6, on tumor vs. paired adjacent nontumor samples from 40 patients with HCC.
Figure 2.
miR-224 expression positively correlates with histone H3 acetylation status in immortalized human liver cells. A) Relative transcript expression of miR-224 and associated Xq28 miRNAs and genes measured using RT-qPCR in immortalized human liver NeHepLxHT cells and human hepatocellular carcinoma HepG2 cells. B) Relative genomic DNA content of Xq28 in NeHepLxHT and HepG2 cells, measured using qPCR. C) Relative transcript expression of miR-224 and associated Xq28 miRNAs and genes in NeHepLxHT (left panel) and HepG2 (right panel) cells treated with 5-Aza (light shaded bars), TSA (dark shaded bars) and SAHA (solid bars), compared to the respective untreated cells (open bars). D) Relative histone H3 acetylation in NeHepLXT cells, measured as relative abundance of chromatin DNA immunoprecipitated with specific antibodies against H3K9 (left panel) and H3K14 (right panel), quantitated with qPCR and normalized against the respective amount of input DNA. E) Relative H3K9 and H3K14 acetylation in NeHepLxHT and HepG2 cells. Data are presented as means ±
se
from ≥3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3.
miR-224 expression is reciprocally regulated by HDAC1, HDAC3, and EP300. A) Relative transcript expression of miR-224 and associated miRNAs and genes in Xq28 when NeHepLxHT cells were transfected with 50 nM of control siRNA (si-Ctrl), individual siRNAs targeting HDAC1 (si-HDAC1), HDAC2 (si-HDAC2), HDAC3 (si-HDAC3), or HDAC8 (si-HDAC8) with miR-16, miR-100, and U6 as unrelated controls. Inset: specific inhibition of each individual HDAC by its intended siRNA. B) Relative transcript expression of miR-224 and associated miRNAs and genes in Xq28 when NeHepLxHT cells were transfected with 50 nM of si-Ctrl, individual siRNAs targeting EP300 (si-EP300), CREBBP (si-CREBBP), or KAT2B (si-KAT2B). Inset: specific inhibition of the intended target genes by the respective siRNAs. C) Relative transcript expression of miR-224 and associated miRNAs and genes in Xq28 when NeHepLxHT cells were transfected with 50 nM of si-Ctrl, si-HDAC1, si-HDAC3, si-EP300, or combination of siRNAs targeting HDAC1 and EP300 (si-HDAC1 and EP300) or HDAC3 and EP300 (si-HDAC3 and EP300). Inset: specific inhibition of the intended target genes by the respective siRNAs. Data are presented as means ±
se
from ≥3 independent experiments. Δ_P_ < 0.05 vs. si-EP300; *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; t test.
Figure 4.
Increased histone H3 acetylation and EP300 binding at the miR-224 associated Xq28 locus in samples from patients with HCC. A) Relative histone H3 acetylation in HCC tumors vs. nontumors, measured as relative abundance of chromatin DNA immunoprecipitated with specific antibodies againstH3K9 (left panel) and H3K14 (right panel), quantitated using qPCR and normalized against the respective amount of input DNA. B) Relative enrichment of HDAC1 (left panel) and EP300 (right panel) binding along Xq28 in HCC tumor vs. nontumor samples. Data are presented as means ±
sd
from 3 patient samples (A, B). C) Relative transcript expression of HDAC1, HDAC3, and EP300 in normal liver (open bars), HCC nontumor tissues (light shaded bars), and HCC tumor (dark shaded bars), measured with RT-qPCR and normalized against β-actin as endogenous control. Data were normalized against the median expression of each respective gene in normal liver samples and presented as box-and-whisker plots with the box spanning the 25th to 75th percentile and the solid line representing the median. D) Scatterplot showing the correlation, expressed as relative fold change, of transcript expression (in log2 scale) between miR-224 and EP300 in tumor vs. paired adjacent nontumor tissues from 28 patients with HCC with significantly high miR-224 expression in tumor (>5-fold higher than the paired adjacent nontumor tissues). Each spot represents data from 1 patient; solid line is the linear regression line. E) Relative transcript expression of EP300, miR-224, and U6 when HepG2 cells were transfected with 50 nM of si-Ctrl or si-EP300. F) Relative transcript expression of miR-224 and U6 when HepG2 cells were treated with specified concentration of EP300-specific inhibitor drug C646. E, F) Data are presented as means ±
se
from ≥3 independent experiments. *P < 0.05. **P < 0.01, ***P < 0.001.
Figure 5.
Schematic diagram illustrating regulation of the chromatin state at the miR-224-associated Xq28 locus by HDAC1, HDAC3, and EP300. In nontransformed liver cells, miR-224 locus is kept in compact transcriptionally quiescent heterochromatin state by HDAC1 and HDAC3. In transformed liver cells, miR-224 is activated through increased histone acetylation mediated by EP300.
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