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. 2008 Jun 20;283(25):17324-32.
doi: 10.1074/jbc.M800224200. Epub 2008 Apr 22.

Enhancer of zeste homologue 2 (EZH2) down-regulates RUNX3 by increasing histone H3 methylation

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Enhancer of zeste homologue 2 (EZH2) down-regulates RUNX3 by increasing histone H3 methylation

Satoshi Fujii et al. J Biol Chem. .

Abstract

Overexpression of enhancer of zeste homologue 2 (EZH2) occurs in various malignancies and is associated with a poor prognosis, especially because of increased cancer cell proliferation. In this study we found an inverse correlation between EZH2 and RUNX3 gene expression in five cancer cell lines, i.e. gastric, breast, prostate, colon, and pancreatic cancer cell lines. Chromatin immunoprecipitation assay showed an association between EZH2 bound to the RUNX3 gene promoter, and trimethylated histone H3 at lysine 27, and HDAC1 (histone deacetylase 1) bound to the RUNX3 gene promoter in cancer cells. RNA interference-mediated knockdown of EZH2 resulted in a decrease in H3K27 trimethylation and unbound HDAC1 and an increase in expression of the RUNX3 gene. Restoration of RUNX3 expression was not associated with any change in DNA methylation status in the RUNX3 promoter region. RUNX3 was repressed by histone deacetylation and hypermethylation of a CpG island in the promoter region and restored by trichostatin A or/and 5-aza-2'-deoxycytidine. Immunofluorescence staining confirmed restoration of expression of the RUNX3 protein after knockdown of EZH2 and its restoration resulted in decreased cell proliferation. In vivo, an inverse relationship between expression of the EZH2 and RUNX3 proteins was observed at the individual cell level in gastric cancer patients in the absence of DNA methylation in the RUNX3 promoter region. The results showed that RUNX3 is a _target for repression by EZH2 and indicated an underlying mechanism of the functional role of EZH2 overexpression on cancer cell proliferation.

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Figures

FIGURE 1.
FIGURE 1.
Restoration of RUNX3 mRNA levels after knockdown of EZH2 in five cancer cell lines, MKN28, MCF-7, LNCap, DLD1, and MiaPaca2. A, the level of EZH2 mRNA after knockdown by siRNA transfection and the restored level of RUNX3 mRNA expression were quantified by real-time RT-PCR. EZH2 mRNA expression levels are shown in a–e, and RUNX3 mRNA expression levels in f–j. E, EZH2 siRNA; C, control siRNA. B, the restored RUNX3 mRNA levels in MKN28 cells were analyzed by RT-PCR and visualized by electrophoresis on 6% polyacrylamide gel.
FIGURE 2.
FIGURE 2.
Restoration of RUNX3 mRNA in gastric and breast cancer cell lines (MKN28 and MCF-7) after EZH2 knockdown accompanied by chromatin remodeling with no change of DNA methylation status in the promoter region of RUNX3 gene. A, a and b, ChIP assay was performed by using the DNA-protein complex isolated from MKN28 and MCF-7 cells transfected with EZH2 siRNA for 96 h and immunoprecipitated with various antibodies, including EZH2 antibody, H3K27me3 antibody (H3-Lys27 trimethylation), HDAC1 antibody, and H3K9me2 antibody (H3-Lys9 dimethylation). The two primer sets for ChIP assay were designated ChIP primer set 1 (a) and ChIP primer set 2 (b). The PCR products of each immunoprecipitated DNA and input DNA were visualized by electrophoresis on a 6% polyacrylamide gel. The number under each gel is the ratio of immunoprecipitated DNA to input DNA quantified (A, antibody; B, no antibody; C, input). c, the MYT1 gene, which is known to be a _target of EZH2, was used to validate ChIP assays in the present study. B, MSP analyses of the DNA of the five cancer cell lines transfected with EZH2 siRNA or negative control siRNA, using primer sets that specifically amplify either methylated DNA(M) or unmethylated DNA (U). Control templates from human genomic placenta DNA treated or untreated with SssI methylase are shown. Ma, 200-bp and 300-bp DNA ladder markers.
FIGURE 3.
FIGURE 3.
Restoration of RUNX3 expression in five cancer cell lines after treatment with 5-aza-dC, TSA, or a combination of 5-aza-dC and TSA. RUNX3 expression in the five lines was evaluated by quantitative real-time RT-PCR. A, MKN28; B, MCF-7; C, LNCap; D, DLD1, and E, MiaPaca2. The concentrations of 5-aza-dC and TSA to which the MCF-7 line was exposed were the same as used by Lau et al. (26). Vehicle, no treatment control; AZA, 5-aza-dC; A+T, combination of 5-aza-dC and TSA.
FIGURE 4.
FIGURE 4.
Knockdown of EZH2-restored RUNX3 expression and decreased cell proliferation by the cancer cell lines. A, RUNX3 re-expression in MKN28 cells was detected by Western blot analysis at 96 h after EZH2 knockdown. B, the nuclear and cytoplasmic distribution of RUNX3 was restored in MKN28 cells transfected with EZH2 siRNA (EZH2, green; RUNX3, red). DRAQ5™ (1,5-bis[[2-(dimethylamino)ethyl]amino]-4,8-dihydroxyanthracene-9,10-dione]) was used to stain the nuclei. C, 48 h after transfection with EZH2 siRNA or control siRNA, cells were re-seeded in new dishes at a density of 1.0 × 105 cells and counted. Points are mean of data from three independent experiments; bars, S.D. Cell proliferation by all five cell lines decreased after transfection with EZH2 siRNA. a, MKN28; b, MCF-7; c, LNCap; d, DLD1; e, MiaPaca2.
FIGURE 5.
FIGURE 5.
DNA methylation of the RUNX3 promoter, and expression of EZH2 protein and RUNX3 protein detected by immunohistochemical staining in tissue specimens from 17 gastric cancers. A, DNA methylation of the RUNX3 promoter was detected by MSP analysis in 11 of the 17 specimens (64.7%). Primer sets that specifically amplified either methylated DNA (M) or unmethylated DNA (U) were the same as used in the MSP analysis of the five cancer cell lines in Fig. 2B. The upper number is the case number. N, non-cancerous tissue; T, tumor tissue. Ma, 200- and 300-bp DNA ladder markers. B and C, histological appearance of the gastric cancer stained with H&E (hematoxylin and eosin) and immunohistochemically for EZH2 and RUNX3. The representative cases are 13 (B) and 2 (C). Immunohistochemical stainings for EZH2 and RUNX3 proteins were performed using two continuous thin tissue sections to show the inverse correlation of expression of EZH2 and RUNX3 proteins in individual cancer cells. The areas of cancer tissue surrounded by square boxes were magnified to show the expression or repression of EZH2 protein and RUNX3 protein of individual cancer cells. In B (case number 13), EZH2 is normally expressed in the germinal center follicular lymphocytes, whereas RUNX3 is normally expressed in the lymphocytes. In B (case number 13), DNA methylation in the RUNX3 promoter was detected in MSP analysis (A), but RUNX3 expression is observed in gastric cancer cells. On the other hand, in C (case number 2), loss of RUNX3 is observed in gastric cancer cells, but no DNA methylation of the promoter region was detected in MSP analysis (A).

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