Extraction of Multiple platforms data
To identify the expression of ZNF132 in BC, a total of 1104 BC tissue samples and 114 normal breast tissue samples that contained the expression of ZNF132 were collected from TCGA database (http://cancergenome.nih.gov/). Additional clinical variables such as age, gender, ER, PR, HER2, metastasis, and clinical stage were analyzed to assess the association between the expression of ZNF132 and these parameters. Other tools, including the Oncomine™ database (www.oncomine.org) and UALCAN platform (http://ualcan.path.uab.edu/) were also used to verify the correlation between ZNF132 expression and clinical outcome, including tissue type, gender, race, clinical stage, molecular pathological characteristics, menstrual status, and survival status.
Clinical samples and immunohistochemical analysis
To detect the expression of ZNF132 in protein level between BC tissue and adjacent normal tissue, we collected 19 cases clinical samples from the First Affiliated Hospital of Xi’an Jiaotong University. These patients did not receive any therapeutic intervention and signed an informed consent before surgery. All patients were finally histologically diagnosed by two pathologists. Ethical approval was provided by the First Affiliated Hospital of Xi’an Jiaotong University Ethics committee. All tissues were fixed in 4% formaldehyde at room temperature for 48h in preparation. The ZNF132 antibody (BIOSS, Beijing, China; cat. no. bs-7150R, 1:1000 dilution) was used for IHC detection. The staining intensity was defined according to the following criterias: staining intensity (negative=0, weak=1, moderate=2, strong=3). Positive staining ratio of ZNF132 (rare: <25%; middle: 25-75%; strong: >75%).
Diagnostic and prognostic significance of ZNF132
The Kaplan-Meier plotter (http://kmplot.com/) was used to assess the prognosis value of ZNF132 in BC patients, including Relapse Free Survival (RFS) and Overall Survival (OS). Besides, univariate and multivariate analysis based on a Cox proportional hazard regression model were performed to evaluate independent prognostic significance of ZNF132, clinical variables included age (≥60 years/<60 years), ER (positive/negative), PR (positive/negative), her2 (positive/negative), tumor size (T2-4/T1), lymph node metastasis (N1-3/N0), distant metastasis (M1/M0), clinical stage (II-IV/I) and expression of ZNF132 (median value). Furthermore, a reciever operating characteristic (ROC) curve was plotted to determine whether the level of ZNF132 expression distinguish the difference between BC tissues and adjacent normal tissues.
To further investigate the molecular mechanisms of ZNF132 in BC, we firstly evaluated the status of ZNF132 that included ZNF132 alteration and its impact on the prognosis of BC patients by the cBioPortal OncoPrint (http://www.cBioPortal.org/index.do). Importantly, Gene Set Enrichment Analysis (GSEA, http://www.linkedomics.org/) was used to predict potential biological processes and pathways. In addition, DNA methylation expression and different methylation sites analysis from the TCGA database were evaluated to identify the downregulated mechanisms of ZNF132 in BC.
To identify the epigenetic effect of ZNF132, 6 human BC cell lines, including MDA-MB-231, MCF7, MDA-MB-453, HCC1937, T47D and DU4475 were used in our study. Each cell line was authenticated by STR analysis in Genesky Co.Ltd (Shanghai, China) and were excluded the mycoplasma contamination using One-step Quickcolor Mycoplasma Detection Kit (Shanghai Yise Medical Technology Co., Ltd.). Then, 2 subgroups were entered into practice, test group was treated with 5µM DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine (5-Aza-dC) (Sigma-Aldrich) and control group was treated with the vehicle. When the cell density was up to 80%, RNA was extracted using TRIzol® protocol. Next, the levels of ZNF132 between test and control group were detected by SYBR®Green FAST (Kapa Biosystems, Inc., Wimington, MA, USA) using Bio-Rad CFX Manager detection system. The PCR protocol followed the parameters: 95˚C for 30 sec, 38 cycles of 5 sec at 95˚C and 30 sec at 55˚C. The primers used are as follow: ZNF132: forward: 5’- CCACAGTGTGATGCTGGAAAACC-3’, reverse: 5’-GCTTTCTTGGTGGAAGGATCTGC-3’; 18s rRNA: forward: 5’-CGCCGCTAGAGGTGAAATTC-3’, reverse: 5’-CTTTCGCTCTGGTCCGTCTT-3’.
In addition, genomic DNA from 6 BC cell lines were extracted according to standard phenol/chloroform protocol. Then, DNA were treated using sodium bisulfite. Briefly, a mixture, including 4μg genomic DNA, 10μg salmon sperm DNA, and 0.3M NaOH, was collected. Next, supply a certain volume of water to a final volume of 20 μl , incubated at 50°C for 20min to denature the DNA. Finally, transfer the mixture into 500μl of solution containing 3M sodium bisulfite and 10mM hydroquinone (Sigma, Saint Louis, MO), incubated at 70°C for 4h. DNA was subsequently purified using the Wizard DNA Clean-Up System (Promega Corp., Madison, WI) and dissovled in distilled water. Importantly, a methylation-specific PCR (MSP) was performed in our study. The PCR procedure was as follows: 4 min denaturation at 95°C, then 45s denaturation at 95°C, 45s anneal at 55°C, and 45s extension at 72°C, repeat this step with 35 cycles, finally an extension at 72°C for 5 min. The reaction products were presented on a 1.2% agarose gel and visualized under UV illumination using an ethidium bromide stain along with a positive control and negative control.
All statistical analyses were performed using SPSS 18.0 (IBM Corp., Armonk, NY, USA) and Graphpad Prism 5.0 software. The association between ZNF132 expression and clinical characteristics was analysed using the Chi-square test. Univariate and Multivariate analyses based on COX regression model were performed to detect connection between clinical variables and the prognosis of BC. Moreover, the ROC curve was identified to evaluate the diagnostic capability between BC and adjacent normal tissue. Student's t-test was used to assess methylation differences of CpG island sites between BC and adjacent normal tissue. P value <0.05 was considered to indicate a statistically significant difference.