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Year : 2018  |  Volume : 55  |  Issue : 4  |  Page : 366--371

Examination of methylation changes of VIM, CXCR4, DOK7, and SPDEF genes in peripheral blood DNA in breast cancer patients

Atefeh Shirkavand, Zahra Niki Boroujeni, Seyed Ahmad Aleyasin 
 Medical Biotechnology Division, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

Correspondence Address:
Seyed Ahmad Aleyasin
Medical Biotechnology Division, National Institute of Genetic Engineering and Biotechnology, Tehran


BACKGROUND: Studying whole blood DNA methylation as a risk marker has valuable applications in either diagnosis or staging of breast cancer. We investigated whole blood DNA methylation status of VIM, CXCR4, DOK7, and SPDEF genes in breast cancer patients in comparison to healthy control subjects. MATERIALS AND METHODS: 60 patients with breast cancer and 40 healthy controls were examined. Genomic DNA isolated from peripheral blood and restriction enzyme polymerase chain reaction (REP) method was applied for analysis. Real-time PCR was used to confirm methylation status of the aforementioned genes and therefore to find out the methylation differences between normal and breast cancer subjects. RESULTS: Level of DOK7 promoter hypomethylation in normal and breast cancer samples was significant (P-value = 0.001). The study, also, showed that hypomethylation of VIM and CXCR4 genes are significant in patients compared with normal cases (P-value < 0.05). Furthermore, SPDEF promoter hypomethylation was not significantly differed between normal and breast cancer samples (P-value = 0.2). CONCLUSIONS: Hypermethylation of DOK7 gene in DNA from patients affected with breast cancer offers a biomarker for diagnosis of the breast cancer. This study indicates that methylation status of VIM and CXCR4 genes changes in breast cancer; so, they can be used as molecular biomarkers in breast cancer prognosis.

How to cite this article:
Shirkavand A, Boroujeni ZN, Aleyasin SA. Examination of methylation changes of VIM, CXCR4, DOK7, and SPDEF genes in peripheral blood DNA in breast cancer patients.Indian J Cancer 2018;55:366-371

How to cite this URL:
Shirkavand A, Boroujeni ZN, Aleyasin SA. Examination of methylation changes of VIM, CXCR4, DOK7, and SPDEF genes in peripheral blood DNA in breast cancer patients. Indian J Cancer [serial online] 2018 [cited 2019 Sep 15 ];55:366-371
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Breast cancer is the most common cancer in Iranian women.[1] There is mounting evidence that epigenetic factors may play a role as a risk factor for breast cancer.[2] Epigenetic is defined as the study of heritable changes in gene expression without any alteration in DNA sequences. Epigenetic changes result in aberrant transcriptional regulation, which lead to changes in the expression pattern of genes. These epigenetic alterations include any changes in DNA methylation profile or histone modifications. DNA methylation plays an important role in the regulation of gene expression in mammalian cells. Aberrant DNA methylation significantly acts in malignancy.[3]

Examination of blood DNA methylation can be used as a biomarker in cancer since the whole blood DNA sampling is noninvasive.[4],[5] Molecular markers are important in staging and the clinical diagnosis of breast cancer.[6] Applying molecular markers for cancer staging and personalized therapy at the time of diagnosis can considerably improve patient care. Whole blood DNA differential methylation status has been linked to risk of some cancers, although little is known for breast cancer.[7],[8] To determine if changes in the level of methylation in DNA derived from peripheral blood cells are related to breast cancer, we analyzed VIM, CXCR4 hypomethylation, and DOK7, SPDEF hypermethylation in the blood of Iranian patients in a case–control study. We also correlated these patterns with clinicopathological data.

The downstream of tyrosine kinase (DOK) protein family has seven members, DOK 1–7. DOK7 is an adaptor protein activates muscle-specific kinase (MuSK).[9] Activated MuSK induces acetylcholine receptor (AChR) that is essential for efficient neuromuscular transmission.[10] In addition to its profound function at neuromuscular junction, a recent study has revealed hypermethylation of DOK7 promoter in primary breast cancer tissues and cell lines. DOK7 plays an inhibitory role on the proliferation and migration of cancer cells in which AKT pathway maybe involved.[11] The precise role of this protein is not entirely clear, although some authors have suggested a potential tumor suppressor role.

The vimentin gene encodes an intermediate filament protein with varied roles in cytoskeletal architecture,[12] immune response,[13] and in collagen mRNA stabilization.[14] Vimentin has a critical role in epithelial-to-mesenchymal transition (EMT) process, including overexpression of other EMT-related genes.[15] Recent studies have identified epigenetic changes in vimentin in solid tumors, such as colorectal, bladder, and breast cancers.[16],[17]

CXCR4 is one of the chemokine receptors involved in cancer progression. The CXCR4 chemokine together with its ligand, CXCL12, involve in the breast cancer metastasis.[18] CXCL12 binding to CXCR4 initiates various signaling pathways that result in increasing intracellular calcium, gene transcription, chemotaxis, cell survival, and proliferation. Previously, hypomethylation of CXCR4 promoter has been recognised in melanoma, breast, and pancreatic cancer.[19],[20]

PDEF (prostate-derived ETS factor) or SPDEF (SAM pointed domain containing ETS transcription factor) is one of the ETS family members that involve in cancer development and progression. Recent studies suggest that SPDEF is a tumor suppressor gene.[21],[22] Hypermethylation of SPDEF promoter has been identified in breast cancer cells.[23]

This study includes a panel of four genes consists of VIM, CXCR4, DOK7, and SPDEF. The aim of this study was to investigate the methylation status of these genes in breast cancer and to determine the association between their methylation profile with clinicopathological features.

 Materials and Methods

Study population

This project was a case–control study. The study group consists of 60 female patients affected with breast cancer who were admitted to Imam Khomeini Hospital in 2016 and 40 control samples obtained from healthy women. The clinicopathological features detailed in [Table 1] including age, stage of disease, and hormone receptor status. All participants gave their written informed consent for research purposes. About 3 mL of whole blood samples were collected from all of them.{Table 1}

Genomic DNA isolation

DNA samples were extracted from whole blood collected in ethylenediaminetetraacetic acid (EDTA) using Roche DNA extraction Kit (Roche Diagnostics, Germany) and stored at 4°C. DNA quality was determined by Nano Drop spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Identifying the CpG islands in VIM, CXCR4, DOK7, and SPDEF genes and designing primers

To determine the methylation status of VIM, CXCR4, DOK7, and SPDEF genes, their related CpG island sequences were identified from CpGPlot/CpGreport website( A typical CpG island in a gene promoter shows ≥60% CpG content and an observed versus expected CpG frequency of ≥0.65 with a high probability of being subject to epigenetic control. These CpG islands span the core promoter and exon1. The CpG islands are located in the promoter region, which is why these ereas are more exposed to methylation. In this study, the sequence of promoters was placed in the CpGPlot/CpGreport software and the best CpG sites were identified. These sites have the following features: 1. GC content of ≥55%, 2. observed/expected CpG ratio of ≥0.65. Restriction endonuclease recognition sites and specific methylation sensitive restriction enzyme obtained by NEBcutter analysis tool. Then, primer sequences designed on either side of selected restriction enzyme recognition site. Designed Primers for amplifying the VIM, CXCR4, DOK7, and SPDEF genes with restriction enzyme polymerase chain reaction (REP) method are shown in [Table 2].{Table 2}

Analysis of promoter methylation with REP method

Detection of hyper or hypomethylated CpG islands in promoter carried out by REP method. Extracted DNA digested using the appropriate methylation-sensitive restriction enzyme [Table 2] according to the manufacturer's instruction. Sac II and Sma I enzymes are sensitive to methylation and methylated regions prevent the enzymes' cleavage. About 50 ng of DNA incubated overnight at 37°C (for complete digestion) with 0.5 μL of restriction enzyme (Takara, Japan), 1-μL 10 × universal buffer and sterilized distilled water to give a final reaction volume of 10 μL. Digested DNA amplified by PCR using DOK7, CXCR4, SPDEF, and VIM-specific primers [Table 2] and the Taq PCR Master Mix Kit (Amplicon, Denmark). An undigested sample containing 0.5 μL nuclease-free water instead of restriction enzyme was employed as a control. PCR performed for 30 cycles using the following thermal cycle conditions: initial denaturation at 95°C for 5 min, denaturation at 95°C for 40 s, primer annealing at 60°C (for CXCR4, DOK7, and SPDEF) or 64°C (for VIM) for 45 s, extension at 72°C for 40 s, and a final extension at 72°C for 7 min. About 20-μL reaction mixture used in PCR, consisting of DNA (50 ng), 0.5 μL of forward and reverse primer (10 pmol/μL), and 10 μL of Taq 2× master mix. PCR products detected using ethidium bromide-stained 1.5% agarose gels and bands visualization took place by UV radiation. The presence of bands with sizes of 300, 304, 301, and 302 bp indicates methylation of CXCR4, DOK7, VIM, and SPDEF promoters, respectively, while the absence of these bands indicates a lack of methylation. The hypomethylation percentage was calculated by gel analyzer software (GelAnalyzer 2010a).

Real-Time PCR

Restriction endonuclease quantitative PCR was applied to confirm methylation alteration in DOK7, CXCR4, SPDEF, and VIM genes. For patient and control samples, treated, and untreated DNA with restriction enzyme (Takara, Japan) put into PCR amplification according to the protocol. All PCRs performed in a Rotor Gene 6000 thermal cycler (Corbett Life Science, Australia). Real-Time PCR accomplished with following conditions: 50 ng of DNA were added to 9 μL of a PCR mixture made up of 5 μL of 2× SYBR Green PCR Master Mix (Takara, Japan), 0.2 μL of each primer, and water. The thermal cycling conditions comprised a denaturation step at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. ΔCt values determined as the difference between the Ct of the treated DNA and the Ct of the untreated DNA.

Statistical analysis

The percentage of promoter methylation changes in patient and normal specimens were analyzed using gel analyzer software (GelAnalyzer 2010a). The real-time PCR data analyzed with LinReg software. The SPSS (version 21) software applied for statistical analysis. Differences in the methylation status of CpG islands in the previously mentioned genes among 60 breast cancer and 40 normal samples was analyzed by t-test. One-way analysis of variance (ANOVA) analyzed association of age, estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2) to the CXCR4, VIM, DOK7, and SPDEF methylation. P value < 0.05 was accepted as a statistically significant.


Association of promotor methylation with clinicopathological parameters

In this study, we analyzed the relation between methylation of four candidate genes including VIM, CXCR4, DOK7, and SPDEF and clinicopathological characters, such as age, stage of cancer, HER2, PR, and ER status. The age range of normal samples was 28–57 years and for the breast group was 32–66 years. Patients with breast cancer were 33.3% HER2+, 66.6% ER+, and 66.6% PR+. Clinical and pathological data of the breast cancer patients are presented in [Table 1]. The patients were categorized into two age groups: <45 and ≥45years. There was not any significant decrease in the status of CXCR4 promoter methylation in older women compared with younger patients (P = 0.346). Similarly, VIM, DOK7, and SPDEF promoter methylation were not associated with age, indicating no dramatic effect of age on methylation status (P = 0.24, P = 0.33, and P = 0.12, respectively). Data analysis in this study indicates promoter methylation of CXCR4 was not significantly associated with ER status (P = 0.33), HER2 expression (P = 0.34), PR status (P = 0.24), and stage of cancer (P = 0.13). Promoter methylation of DOK7 was not significantly associated with ER status (P = 0.11), HER2 expression (P = 0.23), PR status (P = 0.34), and stage of cancer (P = 0.22). Similarly, there was no significant relation between SPDEF promoter methylation and ER status (P = 0.24), HER2 expression (P = 0.32), PR status (P = 0.13), and stage of cancer (P = 0.23). In contrast, methylation of VIM promoter was significantly lower in negative ER specimens (P = 0.002).

Differences in methylation between normal and breast cancer specimens by REP method

In this study, the relationship between promoter methylation patterns of four genes including VIM, CXCR4, DOK7, and SPDEF in peripheral blood DNA of 60 breast cancer patients and 40 normal women was analyzed [Figure 1] and [Figure 2]. Our research indicated VIM and CXCR4 genes are significantly hypomethylated in breast cancer [Figure 1]. In detail, the difference between the percentage of hypomethylation of VIM promoter in normal (18%) and breast cancer samples (63%) was significant (P-value = 0.001) [Figure 3]. Furthermore, CXCR4 promoter was more hypomethylated in breast cancer samples (78%) in comparison to normal samples (48%). DOK7 promoter was hypomethylated in normal samples (65%) compared with breast cancer patients (35%) [Figure 2].{Figure 1}{Figure 2}{Figure 3}

Difference between percentage of hypomethylation of DOK7 promoter in normal and breast cancer samples was significant (P-value = 0.001). Finally, SPDEF promoter was hypomethylated in normal samples (52%) compared with breast cancer patients (45%). Difference between the percentage of hypomethylation of SPDEF promoter in normal and breast cancer samples was not significant (P-value = 0.2).

Real-time PCR

The mean value comparison of ΔCt between normal and patient samples were statistically analyzed using the SPSS software. A significant difference (P value < 0.05) in the methylation status of VIM, DOK7, and CXCR4 genes observed between patient and normal samples [Figure 4] and [Figure 5]. In the case of VIM and CXCR4 genes, the Δ Ct mean value of the control group presented lower levels relative to the patient group. Therefore, gene amplification between digested and undigested patient samples is significant [Figure 4]. However, for DOK7 genes, the Δ Ct mean value of the control group showed a greater amount than the patient group [Figure 5], so that gene amplification between enzyme-treated and enzyme-untreated patient samples is considerable. For SPDEF gene, the Δ Ct mean value of the control group did not show a significant difference (P value > 0.05) with patient samples. Amplification discrepancy indicates differences in the amount of the enzymatic digestion in normal and breast cancer samples, owing to the fact that Sac II and Sma I enzymes are sensitive to methylation; therefore, methylated regions prevent the enzyme's cleavage.{Figure 4}{Figure 5}


Changes in DNA methylation play a critical role in tumorigenesis. Hence, these changes can be used as appropriate biomarkers for diagnosis and prognosis. Blood-based epigenetic biomarkers are being extensively applied. One of the reasons for performing blood test to identify tumors is that blood cells may have undergone epigenetic changes representative of those present in cancer.[24],[25]

In this study, we performed methylation analysis of CpG sites in four genes and identified three genes whose methylation status were significantly associated with breast cancer. These results suggest that the methylation status of peripheral blood DNA, an easily accessible sample, could be used as a biomarker for breast cancer. We used REP method for analyzing of the methylation status of DOK7, CXCR4, VIM, and SPDEF genes.

Promoter hypermethylation of DOK7 implicates as one of the downregulation mechanisms in gene expression.[11] We identified DOK7 promoter was hypomethylated in normal samples (65%) compared with breast cancer patients (35%).

Difference between percentage of hypomethylation of DOK7 promoter in normal and breast cancer samples was significant (P value = 0.001). These data agree with the study of Heyn et al. (2012), suggesting methylation changes in DOK7 as an early event in breast tumorigenesis. They suggested that hypermethylation of DOK7 could be useful as a novel epigenetic blood-based biomarker for this tumor type.[11]

Most breast cancers express higher levels of CXCR4.[26],[27],[28],[29],[30] Holm et al. (2007) and Rhodes et al. (2011) demonstrated that CXCR4 upregulation occurs in many breast cancer patients.[31],[32] Despite many studies on the role of CXCR4 in breast cancer and its epigenetic changes, nevertheless, methylation status of CXCR4 in peripheral blood DNA remained undetermined. In this study, we investigated the methylation status of CXCR4 in Iranian patients and correlated the findings to those in normal women. We identified detectable CXCR4 promoter hypomethylation in the blood from breast cancer patients. Additionally, there was a nonsignificant increase in the CXCR4 promoter methylation in older women compared with younger patients (P = 0.346). Moreover, our research indicates this gene is significantly hypomethylated in breast cancer. The CXCR4 promoter was more hypomethylated in breast cancer samples (78%) than normal samples (48%). Thus, this gene has the potential to be a tumor marker for breast cancer.

Examination of human breast cancer samples showed that vimentin expression predominantly found in carcinomas with low estrogen receptor levels.[33] Our results indicate that VIM gene is significantly hypomethylated in breast cancer; therefore, it can be used as a biomarker for breast cancer samples. Moreover, our research points out there was no relation between the stage of disease, age of patients, (PR, and HER2 statuses. In contrast, methylation of VIM promoter tended to be lower in negative ER specimens and this relation was significant (P = 0.002). These results might have important implications for designing novel therapeutic interventions for breast cancer patients.

Feldman et al. (2003) have suggested that SPDEF levels reduced in invasive breast cancer cells and that re-expression of SPDEF, a tumor suppressor gene, decreases cell migration and invasion.[34] Our research indicates that SPDEF gene is not significantly hypermethylated in blood of breast cancer patients; consequently, it could not be useful as a biomarker in breast cancer. Moreover, our research reveals that there was not any correlation between the methylation of SPDEF gene and the stage of disease, age of patients, ER, PR, and HER2 statuses in breast cancer.

In summary, we confirmed that the hypermethylation of the DOK7 gene in white blood cells' DNA from breast cancer patients can be used as a biomarker for diagnosis of cancer. This study, as well, unveiled changes on the methylation context of the VIM and CXCR4 genes in breast cancer; so, their hypomethylation in blood DNA can be used as a molecular biomarker for breast cancer prognosis.


This study was granted by the National Institute of Genetic Engineering and Biotechnology, Ministry of Science Research and Technology, Tehran, Iran. Dr. Seyed Ahmad Aleyasin is the guarantor of this project and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data.

Financial support and sponsorship

This work was supported by National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

Conflicts of interest

There are no conflicts of interest.


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