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Transcriptional regulation of the p73 gene by Nrf-2 and promoter CpG methylation in human breast cancer.

Lai J, Nie W, Zhang W, Wang Y, Xie R, Wang Y, Gu J, Xu J, Song W, Yang F, Huang G, Cao P, Guan X - Oncotarget (2014)

Bottom Line: However, we found Nrf-2 induced ΔNp73 expression was abolished with 5-aza-dC treatment, thus lead to a down-regulated ΔNp73 and an up-regulated TAp73 expression in breast cancer cells lines.A significant inverse correlation was found between TAp73 and ΔNp73 expression in the above tissue-array (P = 0.047) and validated in another set consisting of 128 breast cancer tumor tissue (P = 0.034).Taken together, our findings suggest that Nrf-2 and promoter methylation cooperatively govern the transcriptional regulation of p73, and unbalanced expression of TAp73 and ΔNp73 expression plays a critical role in breast cancer development.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Jinling Hospital, School of Medicine, Southern Medical University, Guangzhou, China; These authors contributed equally to this work.

ABSTRACT
To understand the transcriptional regulation of p73 by promoter methylation and Nrf-2 in breast carcinogenesis, ChIP assay indicated that Nrf-2 can bind to both promoters and can activate the transcription of TAp73 and ΔNp73 in MCF-7 cell line, knockdown of Nrf-2 gene resulted in an abrogation of TAp73 and ΔNp73 expression in the cells transfected with sh-Nrf-2 as well as Nrf-2 knock out mouse model. However, we found Nrf-2 induced ΔNp73 expression was abolished with 5-aza-dC treatment, thus lead to a down-regulated ΔNp73 and an up-regulated TAp73 expression in breast cancer cells lines. Consistent with this model, we detected decreased TAp73 and increased ΔNp73 expression in breast cancer tissue, along with increased TAp73 but decreased ΔNp73 expression in corresponding surrounding noncancerous tissues (NCTs) in a breast cancer tissue assay. A significant inverse correlation was found between TAp73 and ΔNp73 expression in the above tissue-array (P = 0.047) and validated in another set consisting of 128 breast cancer tumor tissue (P = 0.034). Taken together, our findings suggest that Nrf-2 and promoter methylation cooperatively govern the transcriptional regulation of p73, and unbalanced expression of TAp73 and ΔNp73 expression plays a critical role in breast cancer development.

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Effect of 5-aza-dC on DNMTs activity and Methylation status of P1 and P2 promoters(A) Total DNMT activity was evaluated in MCF-7, SK-BR-3 and MDA-MB-231 after treatment with 0-20μmol/L 5-aza-dC for 48h. Results are the mean remaining DNMT activity ± relative error of three independent experiments. *P < 0.05 or **P < 0.001 vs. untreated controls. (B) The methylation state of the P1 promoter and P2 promoter were detected by Pyrosequencing in MCF-7 after treatment with DMSO (#) or 20μmol/L 5-aza-dC (*) for 48h. Gray columns depict regions of CpG sites, and the percentage methylation at each CpG site is shown on the top. The percentage of methylation is calculated as the C/(C + T) peak ratio per CpG.
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Figure 2: Effect of 5-aza-dC on DNMTs activity and Methylation status of P1 and P2 promoters(A) Total DNMT activity was evaluated in MCF-7, SK-BR-3 and MDA-MB-231 after treatment with 0-20μmol/L 5-aza-dC for 48h. Results are the mean remaining DNMT activity ± relative error of three independent experiments. *P < 0.05 or **P < 0.001 vs. untreated controls. (B) The methylation state of the P1 promoter and P2 promoter were detected by Pyrosequencing in MCF-7 after treatment with DMSO (#) or 20μmol/L 5-aza-dC (*) for 48h. Gray columns depict regions of CpG sites, and the percentage methylation at each CpG site is shown on the top. The percentage of methylation is calculated as the C/(C + T) peak ratio per CpG.

Mentions: To further confirm the nature of the cell death, we used the Annexin V flow cytometry assay to detect the cell apoptosis after the cells were exposed to various concentrations of 5-aza-dC for 48 h (Figure 1C). It has been shown that exposure to 5-aza-dC caused cell apoptosis in a dose-dependent fashion in the three breast cancer cells compared with control, respectively (P<0.05). As shown in Figure 2A, 5-aza-dC treatment inhibited DNMTs activity in breast cancer cells. And pyrosequencing assay successfully detected enrich CPG islands in P1 and P2, and indicated that both P1 and P2 promoters were methylated in breast cancer cell lines, which could be reversed by 5-aza-dC treatment (Figure 2B). In addition, bisulfite sequencing analysis (BSP) was conducted on bisulfite-modified DNA from P2 in MCF-7 cells cultured with 20 μmol/L 5-aza-dC or DMSO (Supplementary Figure S1).


Transcriptional regulation of the p73 gene by Nrf-2 and promoter CpG methylation in human breast cancer.

Lai J, Nie W, Zhang W, Wang Y, Xie R, Wang Y, Gu J, Xu J, Song W, Yang F, Huang G, Cao P, Guan X - Oncotarget (2014)

Effect of 5-aza-dC on DNMTs activity and Methylation status of P1 and P2 promoters(A) Total DNMT activity was evaluated in MCF-7, SK-BR-3 and MDA-MB-231 after treatment with 0-20μmol/L 5-aza-dC for 48h. Results are the mean remaining DNMT activity ± relative error of three independent experiments. *P < 0.05 or **P < 0.001 vs. untreated controls. (B) The methylation state of the P1 promoter and P2 promoter were detected by Pyrosequencing in MCF-7 after treatment with DMSO (#) or 20μmol/L 5-aza-dC (*) for 48h. Gray columns depict regions of CpG sites, and the percentage methylation at each CpG site is shown on the top. The percentage of methylation is calculated as the C/(C + T) peak ratio per CpG.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196172&req=5

Figure 2: Effect of 5-aza-dC on DNMTs activity and Methylation status of P1 and P2 promoters(A) Total DNMT activity was evaluated in MCF-7, SK-BR-3 and MDA-MB-231 after treatment with 0-20μmol/L 5-aza-dC for 48h. Results are the mean remaining DNMT activity ± relative error of three independent experiments. *P < 0.05 or **P < 0.001 vs. untreated controls. (B) The methylation state of the P1 promoter and P2 promoter were detected by Pyrosequencing in MCF-7 after treatment with DMSO (#) or 20μmol/L 5-aza-dC (*) for 48h. Gray columns depict regions of CpG sites, and the percentage methylation at each CpG site is shown on the top. The percentage of methylation is calculated as the C/(C + T) peak ratio per CpG.
Mentions: To further confirm the nature of the cell death, we used the Annexin V flow cytometry assay to detect the cell apoptosis after the cells were exposed to various concentrations of 5-aza-dC for 48 h (Figure 1C). It has been shown that exposure to 5-aza-dC caused cell apoptosis in a dose-dependent fashion in the three breast cancer cells compared with control, respectively (P<0.05). As shown in Figure 2A, 5-aza-dC treatment inhibited DNMTs activity in breast cancer cells. And pyrosequencing assay successfully detected enrich CPG islands in P1 and P2, and indicated that both P1 and P2 promoters were methylated in breast cancer cell lines, which could be reversed by 5-aza-dC treatment (Figure 2B). In addition, bisulfite sequencing analysis (BSP) was conducted on bisulfite-modified DNA from P2 in MCF-7 cells cultured with 20 μmol/L 5-aza-dC or DMSO (Supplementary Figure S1).

Bottom Line: However, we found Nrf-2 induced ΔNp73 expression was abolished with 5-aza-dC treatment, thus lead to a down-regulated ΔNp73 and an up-regulated TAp73 expression in breast cancer cells lines.A significant inverse correlation was found between TAp73 and ΔNp73 expression in the above tissue-array (P = 0.047) and validated in another set consisting of 128 breast cancer tumor tissue (P = 0.034).Taken together, our findings suggest that Nrf-2 and promoter methylation cooperatively govern the transcriptional regulation of p73, and unbalanced expression of TAp73 and ΔNp73 expression plays a critical role in breast cancer development.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Oncology, Jinling Hospital, School of Medicine, Southern Medical University, Guangzhou, China; These authors contributed equally to this work.

ABSTRACT
To understand the transcriptional regulation of p73 by promoter methylation and Nrf-2 in breast carcinogenesis, ChIP assay indicated that Nrf-2 can bind to both promoters and can activate the transcription of TAp73 and ΔNp73 in MCF-7 cell line, knockdown of Nrf-2 gene resulted in an abrogation of TAp73 and ΔNp73 expression in the cells transfected with sh-Nrf-2 as well as Nrf-2 knock out mouse model. However, we found Nrf-2 induced ΔNp73 expression was abolished with 5-aza-dC treatment, thus lead to a down-regulated ΔNp73 and an up-regulated TAp73 expression in breast cancer cells lines. Consistent with this model, we detected decreased TAp73 and increased ΔNp73 expression in breast cancer tissue, along with increased TAp73 but decreased ΔNp73 expression in corresponding surrounding noncancerous tissues (NCTs) in a breast cancer tissue assay. A significant inverse correlation was found between TAp73 and ΔNp73 expression in the above tissue-array (P = 0.047) and validated in another set consisting of 128 breast cancer tumor tissue (P = 0.034). Taken together, our findings suggest that Nrf-2 and promoter methylation cooperatively govern the transcriptional regulation of p73, and unbalanced expression of TAp73 and ΔNp73 expression plays a critical role in breast cancer development.

Show MeSH
Related in: MedlinePlus