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Zinc-finger protein 545 inhibits cell proliferation as a tumor suppressor through inducing apoptosis and is disrupted by promoter methylation in breast cancer.

Xiao Y, Xiang T, Luo X, Li C, Li Q, Peng W, Li L, Li S, Wang Z, Tang L, Ren G, Tao Q - PLoS ONE (2014)

Bottom Line: In this study, we found that ZNF545 was frequently downregulated in estrogen receptor-positive (ER+), progesterone receptor-positive (PR+) and human epidermal growth factor receptor 2-negative (HER2-) breast tumor tissues compared with paired adjacent non-tumor tissues.We found that ZNF545 was silenced by promoter methylation in MCF7 cell line, and its expression could be restored by demethylation, concomitant with increased unmethylated alleles.Taken together, these results demonstrate that ZNF545 inhibits breast tumor cell proliferation through inducing apoptosis and is disrupted by promoter methylation in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.

ABSTRACT
Krüppel-associated box-containing zinc finger proteins (KRAP-ZFPs) are well recognized as key regulators of transcription, which play a crucial role in the regulation of cell proliferation, differentiation, apoptosis and tumorigenesis. We previously identified a KRAP-ZFP protein ZNF545 acting as a tumor suppressor involved in tumor pathogenesis. However, its expression and biological function in breast cancer remain elusive. In this study, we found that ZNF545 was frequently downregulated in estrogen receptor-positive (ER+), progesterone receptor-positive (PR+) and human epidermal growth factor receptor 2-negative (HER2-) breast tumor tissues compared with paired adjacent non-tumor tissues. We further examined its expression and methylation in breast cancer cell lines by semi-quantitative RT-PCR and methylation-specific PCR. We found that ZNF545 was silenced by promoter methylation in MCF7 cell line, and its expression could be restored by demethylation, concomitant with increased unmethylated alleles. ZNF545 methylation was detected in 29% of breast tumor tissues, but not in normal breast tissues, suggesting tumor-specific methylation of ZNF545 in breast cancer. Ectopic expression of ZNF545 in MCF7 cells inhibited cell proliferation through inducing cell cycle G0/G1 arrest and apoptosis, thus as a tumor suppressor. Moreover, ZNF545 upregulated mRNA and protein levels of c-Jun/AP1, BAX, p53 and Caspase 3. Taken together, these results demonstrate that ZNF545 inhibits breast tumor cell proliferation through inducing apoptosis and is disrupted by promoter methylation in breast cancer.

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Ectopic expression of ZNF545 induced apoptosis and cell cycle arrest in MCF7 cells.(A) Anlysis of cell cycle distribution of vector-, ZNF545-transfected MCF7 cells. The distribution and percentage of cells in G1, S and G2/M phase of the cell cycle are indicated. B) Representative flow cytometry histograms of cell cycle alterations. The assay was performed in triplicate (*, P<0.05).(C), Apoptosis was examined using Acridine orange/ethidium bromide (AO/EB) fluorescence staining. The percentage of apoptotic cells is indicated(***, P<0.001). (D). Expression levels of C-Jun, BAX, p53 and Casp3 were evaluated by real-time PCR in vector-, ZNF545-transfected MCF7 cells. (E). Expression levels of C-Jun, BAX, p53 and Casp3 were evaluated by westernblot in vector-, ZNF545-transfected MCF7 cells.
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pone-0110990-g004: Ectopic expression of ZNF545 induced apoptosis and cell cycle arrest in MCF7 cells.(A) Anlysis of cell cycle distribution of vector-, ZNF545-transfected MCF7 cells. The distribution and percentage of cells in G1, S and G2/M phase of the cell cycle are indicated. B) Representative flow cytometry histograms of cell cycle alterations. The assay was performed in triplicate (*, P<0.05).(C), Apoptosis was examined using Acridine orange/ethidium bromide (AO/EB) fluorescence staining. The percentage of apoptotic cells is indicated(***, P<0.001). (D). Expression levels of C-Jun, BAX, p53 and Casp3 were evaluated by real-time PCR in vector-, ZNF545-transfected MCF7 cells. (E). Expression levels of C-Jun, BAX, p53 and Casp3 were evaluated by westernblot in vector-, ZNF545-transfected MCF7 cells.

Mentions: Flow cytometric analysis of cell cycle and AO/EB staining were used to assess the mechanism of ZNF545 in inhibiting cell proliferation. ZNF545 obviously increased the number of MCF7 cells in the G0-G1 phase from 47.42% to 52.36%(*p<0.05) compared to controls (Fig. 4A, B). These results suggested that cell proliferation inhibition by ZNF545 is likely mediated through cell cycle arrest at G0/G1.


Zinc-finger protein 545 inhibits cell proliferation as a tumor suppressor through inducing apoptosis and is disrupted by promoter methylation in breast cancer.

Xiao Y, Xiang T, Luo X, Li C, Li Q, Peng W, Li L, Li S, Wang Z, Tang L, Ren G, Tao Q - PLoS ONE (2014)

Ectopic expression of ZNF545 induced apoptosis and cell cycle arrest in MCF7 cells.(A) Anlysis of cell cycle distribution of vector-, ZNF545-transfected MCF7 cells. The distribution and percentage of cells in G1, S and G2/M phase of the cell cycle are indicated. B) Representative flow cytometry histograms of cell cycle alterations. The assay was performed in triplicate (*, P<0.05).(C), Apoptosis was examined using Acridine orange/ethidium bromide (AO/EB) fluorescence staining. The percentage of apoptotic cells is indicated(***, P<0.001). (D). Expression levels of C-Jun, BAX, p53 and Casp3 were evaluated by real-time PCR in vector-, ZNF545-transfected MCF7 cells. (E). Expression levels of C-Jun, BAX, p53 and Casp3 were evaluated by westernblot in vector-, ZNF545-transfected MCF7 cells.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215924&req=5

pone-0110990-g004: Ectopic expression of ZNF545 induced apoptosis and cell cycle arrest in MCF7 cells.(A) Anlysis of cell cycle distribution of vector-, ZNF545-transfected MCF7 cells. The distribution and percentage of cells in G1, S and G2/M phase of the cell cycle are indicated. B) Representative flow cytometry histograms of cell cycle alterations. The assay was performed in triplicate (*, P<0.05).(C), Apoptosis was examined using Acridine orange/ethidium bromide (AO/EB) fluorescence staining. The percentage of apoptotic cells is indicated(***, P<0.001). (D). Expression levels of C-Jun, BAX, p53 and Casp3 were evaluated by real-time PCR in vector-, ZNF545-transfected MCF7 cells. (E). Expression levels of C-Jun, BAX, p53 and Casp3 were evaluated by westernblot in vector-, ZNF545-transfected MCF7 cells.
Mentions: Flow cytometric analysis of cell cycle and AO/EB staining were used to assess the mechanism of ZNF545 in inhibiting cell proliferation. ZNF545 obviously increased the number of MCF7 cells in the G0-G1 phase from 47.42% to 52.36%(*p<0.05) compared to controls (Fig. 4A, B). These results suggested that cell proliferation inhibition by ZNF545 is likely mediated through cell cycle arrest at G0/G1.

Bottom Line: In this study, we found that ZNF545 was frequently downregulated in estrogen receptor-positive (ER+), progesterone receptor-positive (PR+) and human epidermal growth factor receptor 2-negative (HER2-) breast tumor tissues compared with paired adjacent non-tumor tissues.We found that ZNF545 was silenced by promoter methylation in MCF7 cell line, and its expression could be restored by demethylation, concomitant with increased unmethylated alleles.Taken together, these results demonstrate that ZNF545 inhibits breast tumor cell proliferation through inducing apoptosis and is disrupted by promoter methylation in breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Epigenetics Laboratory, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China; Department of Endocrine and Breast Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.

ABSTRACT
Krüppel-associated box-containing zinc finger proteins (KRAP-ZFPs) are well recognized as key regulators of transcription, which play a crucial role in the regulation of cell proliferation, differentiation, apoptosis and tumorigenesis. We previously identified a KRAP-ZFP protein ZNF545 acting as a tumor suppressor involved in tumor pathogenesis. However, its expression and biological function in breast cancer remain elusive. In this study, we found that ZNF545 was frequently downregulated in estrogen receptor-positive (ER+), progesterone receptor-positive (PR+) and human epidermal growth factor receptor 2-negative (HER2-) breast tumor tissues compared with paired adjacent non-tumor tissues. We further examined its expression and methylation in breast cancer cell lines by semi-quantitative RT-PCR and methylation-specific PCR. We found that ZNF545 was silenced by promoter methylation in MCF7 cell line, and its expression could be restored by demethylation, concomitant with increased unmethylated alleles. ZNF545 methylation was detected in 29% of breast tumor tissues, but not in normal breast tissues, suggesting tumor-specific methylation of ZNF545 in breast cancer. Ectopic expression of ZNF545 in MCF7 cells inhibited cell proliferation through inducing cell cycle G0/G1 arrest and apoptosis, thus as a tumor suppressor. Moreover, ZNF545 upregulated mRNA and protein levels of c-Jun/AP1, BAX, p53 and Caspase 3. Taken together, these results demonstrate that ZNF545 inhibits breast tumor cell proliferation through inducing apoptosis and is disrupted by promoter methylation in breast cancer.

Show MeSH
Related in: MedlinePlus