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Epigenetic alterations of the keratin 13 gene in oral squamous cell carcinoma.

Naganuma K, Hatta M, Ikebe T, Yamazaki J - BMC Cancer (2014)

Bottom Line: ChIP analysis revealed that PRC2-mediated trimethylation of Lys 27 on histone H3 (H3K27me3) was increased in the KRT13 promoter in the HSC3 and SAS cells.Finally, we demonstrated that the treatment of SAS cells with DZNep reactivated the transcription of KRT13 gene.Our data provide mechanistic insights into the epigenetic silencing of KRT13 genes in OSCC cells and might be useful for the development of diagnostic markers and novel therapeutic approaches against OSCCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka 814-0193, Japan. hatta@college.fdcnet.ac.jp.

ABSTRACT

Background: Epigenetic modifications play important roles in the regulation of gene expression determining cellular phenotype as well as various pathologies such as cancer. Although the loss of keratin 13 (KRT13) is reportedly linked to malignant transformation of oral epithelial cells, the molecular mechanisms through which KRT13 is repressed in oral squamous cell carcinoma (OSCC) remain unclear. The aim of this study is to identify the epigenetic alterations of the KRT13 gene in OSCCs.

Methods: We investigated KRT13 expression levels and chromatin modifications of the KRT13 promoter in the three OSCC cell lines (HSC4, HSC3, and SAS). The expression levels of KRT13 protein and mRNA were analyzed by western blotting and quantitative reverse-transcription polymerase chain reaction, respectively, and the localization of KRT13 protein was detected by immunofluorescence. DNA methylation and histone modifications in the KRT13 promoter were determined by bisulfite sequencing and chromatin immunoprecipitation (ChIP), respectively. For the pharmacological depletion of Polycomb repressive complex 2 (PRC2), cells were treated with 3-deazaneplanocin A (DZNep).

Results: KRT13 expression was transcriptionally silenced in the HSC3 and SAS cells and post-transcriptionally repressed in the HSC4 cells, while the KRT13 promoter was hypermethylated in all of the three OSCC cell lines. ChIP analysis revealed that PRC2-mediated trimethylation of Lys 27 on histone H3 (H3K27me3) was increased in the KRT13 promoter in the HSC3 and SAS cells. Finally, we demonstrated that the treatment of SAS cells with DZNep reactivated the transcription of KRT13 gene.

Conclusions: Our data provide mechanistic insights into the epigenetic silencing of KRT13 genes in OSCC cells and might be useful for the development of diagnostic markers and novel therapeutic approaches against OSCCs.

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Related in: MedlinePlus

Effects of 3-deazaneplanocin A (DZNep) on KRT13 expression in OSCC cells. (A) SAS cells were treated with 10 μM DZNep for 24 h, and the levels of the PRC2 proteins (Ezh2 and Suz12) and H3K27me3 were examined by western blotting. Fold change in PRC2 proteins and H3K27me3 were normalized to β-actin and total histone H3, respectively, and calculated relative to band intensity of the control condition samples. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student’s t-test. **p < 0.01. (B) KRT13 mRNA levels were analyzed by quantitative RT-PCR. Fold change in KRT13 mRNA was normalized to GAPDH and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student’s t-test. **p < 0.01. (C) H3K27me3 levels in the KRT13 promoter were analyzed by ChIP assay. Fold enrichment of histone H3K27me3 was normalized to that of total histone H3 and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3–5) are shown. Statistical analysis was performed using Student’s t-test. *p < 0.05.
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Fig5: Effects of 3-deazaneplanocin A (DZNep) on KRT13 expression in OSCC cells. (A) SAS cells were treated with 10 μM DZNep for 24 h, and the levels of the PRC2 proteins (Ezh2 and Suz12) and H3K27me3 were examined by western blotting. Fold change in PRC2 proteins and H3K27me3 were normalized to β-actin and total histone H3, respectively, and calculated relative to band intensity of the control condition samples. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student’s t-test. **p < 0.01. (B) KRT13 mRNA levels were analyzed by quantitative RT-PCR. Fold change in KRT13 mRNA was normalized to GAPDH and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student’s t-test. **p < 0.01. (C) H3K27me3 levels in the KRT13 promoter were analyzed by ChIP assay. Fold enrichment of histone H3K27me3 was normalized to that of total histone H3 and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3–5) are shown. Statistical analysis was performed using Student’s t-test. *p < 0.05.

Mentions: To determine whether PRC2-mediated transcriptional repression is responsible for KRT13 gene silencing, we examined the effects of the potent PCR2 inhibitor DZNep on KRT13 mRNA expression in the SAS cells. As shown by western blotting (Figure 5A), treatment of the SAS cells with 10 μM DZNep for 24 h decreased the Ezh2 and Suz12 protein levels and H3K27 trimethylation levels, and KRT13 mRNA expression was markedly increased by almost 30-fold after DZNep treatment (Figure 5B). In addition, the H3K27me3 levels in the KRT13 promoter were reduced in the SAS cells treated with DZNep (Figure 5C). We then investigated the reversibility of DZNep-induced KRT13 transcription in SAS cells, which had been treated with DZNep for 24 h. KRT13 mRNA levels were increased after additional exposure to DZNep for 48 h, whereas the mRNA levels were reversed for 48 h after withdrawing DZNep (Figure 6A). As shown in Figure 6B, the Ezh2 and Suz12 protein levels were decreased by DZNep treatment, and increased more than normal levels after drug removal. Taken together, our data suggest that DZNep induces the down-regulation of Ezh2 proteins and reactivates the transcription of KRT13 gene in SAS cells and that the effects of DZNep treatment on SAS cells are not heritable changes.Figure 5


Epigenetic alterations of the keratin 13 gene in oral squamous cell carcinoma.

Naganuma K, Hatta M, Ikebe T, Yamazaki J - BMC Cancer (2014)

Effects of 3-deazaneplanocin A (DZNep) on KRT13 expression in OSCC cells. (A) SAS cells were treated with 10 μM DZNep for 24 h, and the levels of the PRC2 proteins (Ezh2 and Suz12) and H3K27me3 were examined by western blotting. Fold change in PRC2 proteins and H3K27me3 were normalized to β-actin and total histone H3, respectively, and calculated relative to band intensity of the control condition samples. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student’s t-test. **p < 0.01. (B) KRT13 mRNA levels were analyzed by quantitative RT-PCR. Fold change in KRT13 mRNA was normalized to GAPDH and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student’s t-test. **p < 0.01. (C) H3K27me3 levels in the KRT13 promoter were analyzed by ChIP assay. Fold enrichment of histone H3K27me3 was normalized to that of total histone H3 and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3–5) are shown. Statistical analysis was performed using Student’s t-test. *p < 0.05.
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Fig5: Effects of 3-deazaneplanocin A (DZNep) on KRT13 expression in OSCC cells. (A) SAS cells were treated with 10 μM DZNep for 24 h, and the levels of the PRC2 proteins (Ezh2 and Suz12) and H3K27me3 were examined by western blotting. Fold change in PRC2 proteins and H3K27me3 were normalized to β-actin and total histone H3, respectively, and calculated relative to band intensity of the control condition samples. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student’s t-test. **p < 0.01. (B) KRT13 mRNA levels were analyzed by quantitative RT-PCR. Fold change in KRT13 mRNA was normalized to GAPDH and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3) are shown. Statistical analysis was performed using Student’s t-test. **p < 0.01. (C) H3K27me3 levels in the KRT13 promoter were analyzed by ChIP assay. Fold enrichment of histone H3K27me3 was normalized to that of total histone H3 and calculated relative to that of the cells under the control condition. The means ± SEM for each group (n = 3–5) are shown. Statistical analysis was performed using Student’s t-test. *p < 0.05.
Mentions: To determine whether PRC2-mediated transcriptional repression is responsible for KRT13 gene silencing, we examined the effects of the potent PCR2 inhibitor DZNep on KRT13 mRNA expression in the SAS cells. As shown by western blotting (Figure 5A), treatment of the SAS cells with 10 μM DZNep for 24 h decreased the Ezh2 and Suz12 protein levels and H3K27 trimethylation levels, and KRT13 mRNA expression was markedly increased by almost 30-fold after DZNep treatment (Figure 5B). In addition, the H3K27me3 levels in the KRT13 promoter were reduced in the SAS cells treated with DZNep (Figure 5C). We then investigated the reversibility of DZNep-induced KRT13 transcription in SAS cells, which had been treated with DZNep for 24 h. KRT13 mRNA levels were increased after additional exposure to DZNep for 48 h, whereas the mRNA levels were reversed for 48 h after withdrawing DZNep (Figure 6A). As shown in Figure 6B, the Ezh2 and Suz12 protein levels were decreased by DZNep treatment, and increased more than normal levels after drug removal. Taken together, our data suggest that DZNep induces the down-regulation of Ezh2 proteins and reactivates the transcription of KRT13 gene in SAS cells and that the effects of DZNep treatment on SAS cells are not heritable changes.Figure 5

Bottom Line: ChIP analysis revealed that PRC2-mediated trimethylation of Lys 27 on histone H3 (H3K27me3) was increased in the KRT13 promoter in the HSC3 and SAS cells.Finally, we demonstrated that the treatment of SAS cells with DZNep reactivated the transcription of KRT13 gene.Our data provide mechanistic insights into the epigenetic silencing of KRT13 genes in OSCC cells and might be useful for the development of diagnostic markers and novel therapeutic approaches against OSCCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka 814-0193, Japan. hatta@college.fdcnet.ac.jp.

ABSTRACT

Background: Epigenetic modifications play important roles in the regulation of gene expression determining cellular phenotype as well as various pathologies such as cancer. Although the loss of keratin 13 (KRT13) is reportedly linked to malignant transformation of oral epithelial cells, the molecular mechanisms through which KRT13 is repressed in oral squamous cell carcinoma (OSCC) remain unclear. The aim of this study is to identify the epigenetic alterations of the KRT13 gene in OSCCs.

Methods: We investigated KRT13 expression levels and chromatin modifications of the KRT13 promoter in the three OSCC cell lines (HSC4, HSC3, and SAS). The expression levels of KRT13 protein and mRNA were analyzed by western blotting and quantitative reverse-transcription polymerase chain reaction, respectively, and the localization of KRT13 protein was detected by immunofluorescence. DNA methylation and histone modifications in the KRT13 promoter were determined by bisulfite sequencing and chromatin immunoprecipitation (ChIP), respectively. For the pharmacological depletion of Polycomb repressive complex 2 (PRC2), cells were treated with 3-deazaneplanocin A (DZNep).

Results: KRT13 expression was transcriptionally silenced in the HSC3 and SAS cells and post-transcriptionally repressed in the HSC4 cells, while the KRT13 promoter was hypermethylated in all of the three OSCC cell lines. ChIP analysis revealed that PRC2-mediated trimethylation of Lys 27 on histone H3 (H3K27me3) was increased in the KRT13 promoter in the HSC3 and SAS cells. Finally, we demonstrated that the treatment of SAS cells with DZNep reactivated the transcription of KRT13 gene.

Conclusions: Our data provide mechanistic insights into the epigenetic silencing of KRT13 genes in OSCC cells and might be useful for the development of diagnostic markers and novel therapeutic approaches against OSCCs.

Show MeSH
Related in: MedlinePlus