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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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Effects of ZNF545 on colony formation and cell proliferation of MCF7cells.(A) Representative colony formation assay and Quantitative analysis of colony formation. The numbers of G418-resistant colonies in vector-transfected controls were set to 100%, Values are expressed as the mean±SD from three experiments, and the asterisk indicates the statistical significance compared to the controls (***, p<0.001). (B) Expression of ZNF545 by RT-PCR in vector- and ZNF545-transfected MCF-7 cells. (C) CCK-8 assay for cell proliferation on vector- and ZNF545-transfecetd MCF7 cells. Asterisks indicate a significant level of proliferation compared with controls (**, p<0.01).
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pone-0110990-g003: Effects of ZNF545 on colony formation and cell proliferation of MCF7cells.(A) Representative colony formation assay and Quantitative analysis of colony formation. The numbers of G418-resistant colonies in vector-transfected controls were set to 100%, Values are expressed as the mean±SD from three experiments, and the asterisk indicates the statistical significance compared to the controls (***, p<0.001). (B) Expression of ZNF545 by RT-PCR in vector- and ZNF545-transfected MCF-7 cells. (C) CCK-8 assay for cell proliferation on vector- and ZNF545-transfecetd MCF7 cells. Asterisks indicate a significant level of proliferation compared with controls (**, p<0.01).

Mentions: To further determine whether ZNF545 is a functional TSG in breast cancer, the effect of ZNF545 on MCF7 cell proliferation was examined by colony formation assay and CCK8 assay. Results showed that ZNF545 markedly reduced the efficiency of MCF7 colony formation to ∼10% compared to controls (***p<0.001) (Fig. 3A). RT-PCR confirmed ZNF545 expression in ZNF545-transfected MCF cells (Fig. 3B). After transfection with ZNF545 in MCF7 cells, cell viability significantly decreased at 24 h, 48 h and 72 h (**p<0.01) (Fig. 3C).


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)

Effects of ZNF545 on colony formation and cell proliferation of MCF7cells.(A) Representative colony formation assay and Quantitative analysis of colony formation. The numbers of G418-resistant colonies in vector-transfected controls were set to 100%, Values are expressed as the mean±SD from three experiments, and the asterisk indicates the statistical significance compared to the controls (***, p<0.001). (B) Expression of ZNF545 by RT-PCR in vector- and ZNF545-transfected MCF-7 cells. (C) CCK-8 assay for cell proliferation on vector- and ZNF545-transfecetd MCF7 cells. Asterisks indicate a significant level of proliferation compared with controls (**, p<0.01).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110990-g003: Effects of ZNF545 on colony formation and cell proliferation of MCF7cells.(A) Representative colony formation assay and Quantitative analysis of colony formation. The numbers of G418-resistant colonies in vector-transfected controls were set to 100%, Values are expressed as the mean±SD from three experiments, and the asterisk indicates the statistical significance compared to the controls (***, p<0.001). (B) Expression of ZNF545 by RT-PCR in vector- and ZNF545-transfected MCF-7 cells. (C) CCK-8 assay for cell proliferation on vector- and ZNF545-transfecetd MCF7 cells. Asterisks indicate a significant level of proliferation compared with controls (**, p<0.01).
Mentions: To further determine whether ZNF545 is a functional TSG in breast cancer, the effect of ZNF545 on MCF7 cell proliferation was examined by colony formation assay and CCK8 assay. Results showed that ZNF545 markedly reduced the efficiency of MCF7 colony formation to ∼10% compared to controls (***p<0.001) (Fig. 3A). RT-PCR confirmed ZNF545 expression in ZNF545-transfected MCF cells (Fig. 3B). After transfection with ZNF545 in MCF7 cells, cell viability significantly decreased at 24 h, 48 h and 72 h (**p<0.01) (Fig. 3C).

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