Identification Of Zinc-finger Protein545as A Tumor Suppressor And Methylation In Breast Cancers

ObjectiveThe aim of this study was to examine expression and methylation ofZNF545in breast cancer, including breast cancer cells and paired breasttumor tissues, to analyze the relationship of ZNF545methylation withclinicopathological features of breast cancer patients, to determine thebiological function of ZNF545and the mechanism in breast cancers.Methods1. ZNF545expression in six breast cancer cell lines （BT549,MDA-MB-231, MDA-MB468, MCF-7, T47D, SK-BR-3）and paired breasttumor tissues was detected by reverse transcriptase–polymerase chainreaction and quantitative real-time PCR, respectively.2. We analyzed the online microarray database (Oncomine, CompendiaBioscience,Ann Arbor, MI), to exam ZNF545expression in breast tumortissues.3. ZNF545methylation in breast cancer cell lines and paired breasttumor tissues was detected by Bisulfite modification of DNA and methylation-specific PCR. The relationship of ZNF545methylation withclinicopathological features of breast cancer patients was further analyzed.4. To determine ZNF545function in breast cancer, the effect of ZNF545on MCF7cell proliferation was examined by colony formation assay andCCK8assay.5. Flow cytometric analysis of cell cycle and AO/EB staining were usedto assess the mechanism of ZNF545in inhibiting cell proliferation.Results1. We found that ZNF545was downregulated in70%(14/20) of breastcancer tissues, compared with their paired adjacent non-tumor tissues, andwas silenced in MCF7cells.2. ZNF545was significantly downregulated in breast cancer, throughanalyzing the online microarray database (Oncomine, CompendiaBioscience,Ann Arbor, MI).3. We found that ZNF545was silenced by promoter methylation inMCF7cell line, and its expression could be restored by demethylation,concomitant with increased unmethylated alleles. ZNF545methylation wasdetected in29%of breast tumor tissues, but not in normal breast tissues.4. ZNF545markedly reduced the efficiency of MCF7colony formationto~10%compared to controls (p<0.05). After transfection with ZNF545inMCF7cells, cell viability significantly decreased at24h,48h and72h(p<0.05). 5. ZNF545obviously increased the number of MCF7cells in the G0-G1phase from47.42%to52.36%(p<0.05) compared to controls and inducedapoptosis in MCF7cells (p<0.05). ZNF545-expressing MCF7cells byqRT-PCR, and found that ZNF545could upregulate expression ofc-Jun/AP1, BAX, p53and Caspase3in mRNA levelConclusionWe found that ZNF545was silenced by promoter methylation in MCF7cell line, and its expression could be restored by demethylation, concomitantwith increased unmethylated alleles. ZNF545methylation was detected in29%of breast tumor tissues, but not in normal breast tissues, suggestingtumor-specific methylation of ZNF545in breast cancer. Ectopic expressionof ZNF545in MCF7cells inhibited cell proliferation through inducing cellcycle G0/G1arrest and apoptosis, thus as a tumor suppressor. Moreover,ZNF545upregulated mRNA levels of c-Jun/AP1, BAX, p53and Caspase3.Taken together, these results demonstrate that ZNF545inhibits breast tumorcell proliferation through inducing apoptosis and is disrupted by promotermethylation in breast cancer.