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Disease Markers
Volume 2016 (2016), Article ID 5780538, 7 pages
http://dx.doi.org/10.1155/2016/5780538
Research Article

Detection of OSR2, VAV3, and PPFIA3 Methylation in the Serum of Patients with Gastric Cancer

1The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China
2No. 2 Subsidiary Hospital of No. 4 Military Medical University, Xi’an, Shaanxi, China

Received 28 December 2015; Revised 19 February 2016; Accepted 2 March 2016

Academic Editor: Stamatios Theocharis

Copyright © 2016 Wen-han Li et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Aim. This study was to evaluate the diagnostic value of OSR2, VAV3, and PPFIA3 hypermethylation in gastric cancer (GC) patients. Patients and Methods. By using methylation-specific polymerase chain reaction (MSP), we detected the methylation status in tissue and serum samples from 48 gastric cancer (GC) patients and 25 normal individuals. Results. We found that OSR2, VAV3, and PPFIA3 were methylated in 70.8% (34/48), 54.2% (26/48), and 60.4% (29/48) of GC tissue, respectively. On the contrary, those genes were barely methylated in their paired paracancerous histological normal tissues (PCHNTs) (all values < 0.01). We next analyzed the methylated OSR2, VAV3, and PPFIA3 in serum DNA. Compared with 25 normal individuals, those three genes were significantly hypermethylated in GC patients serum samples (all values < 0.01). Regarding their diagnostic value in serum samples, the combined sensitivity of at least one positive among the three markers in serum was 83.3%, with a specificity of 88%. Conclusion. Our test suggested that methylation of OSR2, VAV3, and PPFIA3 genes in serum sample may offer a good alternative in a simple, promising, and noninvasive detection of GC.

1. Introduction

Although the incidence of gastric cancer (GC) has declined in the past decades, it is still one of the most common malignancies worldwide. According to related data, the annual diagnosis number of GC approximately reaches one million; among them 42% are from China [1, 2]. As GC is usually asymptomatic until advanced stage, it is often associated with poor treatment outcome and low 5-year survival rate. Therefore, an ideal screening tool to detect GC with high sensitivity and specificity has a high priority. Upper endoscopy can distinguish between cancerous and noncancerous conditions by performing biopsies of suspicious areas, therefore being considered as the golden standard for GC detection. However, considering its invasive operations, high-risk patients of GC are reluctant to take this approach as a regular examination. Serum tumor markers, such as carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, and carbohydrate antigen (CA) 724, have been widely applied in clinical practice. However, none of them are suitable for early detection of GC. Till now, there is no ideal diagnostic method with relatively high sensitivity that could be applied in clinical screening for GC.

As we all know, GC is a multistep process and accumulating data have elucidated that epigenetic alterations, especially DNA methylation, play an important role in GC initiation. By silencing the tumor suppressor genes which play a key role in DNA repair, cell adhesion, cell cycle control, and apoptosis [3], DNA hypermethylation at CpG islands in or near promoter regions contributes a lot during the process of carcinogenesis. Moreover, it has been shown that changes of methylation in body fluids paralleled other somatic tissues and therefore are thought to be connected with certain cancers [4, 5]. On the basis of those studies above, many attempts have been made to investigate biomarkers in serum, urine, and sputum in various malignancies [611]. As for GC, many researchers thought serum DNA-based technique is a promising alternative for its relatively noninvasive operations as well as a convenient tissue to assay for constitutional methylation. Many studies have been made to explore the feasibility of the use of serum as a biomarker for certain cancers [4, 5, 1218].

The selection of the candidate gene for analyzing is crucial to improving the sensitivity and specificity of methylation DNA test. Current microarray technology provides us with an opportunity for high-throughput unbiased methylation analysis of a large number of CpG sites [19]. By using an Infinium HumanMethylation 450 BeadChip array, Zong and colleagues [20] identified three genes (OSR2, VAV3, and PPFIA3) that were hypermethylated in GC tissue. Odd-skipped related 2 (OSR2), which contains DNA-binding C2H2-type zinc finger domains in the C-terminal half, plays an important role in cellular quiescence and proliferation under epigenetic regulation [21, 22]. Additionally, recent articles have reported that VAV3 (a member of VAV gene family which plays an important role in the process of tumor development and metastasis) and PTPRF-Interacting Protein Alpha-3 (PPFIA3) may associated with the tumorigenesis and development of GC [20, 23]. In the present study, we sought to explore the feasibility of DNA methylation status of OSR2, VAV3, and PPFIA3 as a noninvasive screening tool for GC.

2. Materials and Methods

2.1. Collection of Tissue and Peripheral Blood Samples

In order to reduce bias, we designed this experiment as a blinded assay. All sample collection and preservation were taken care of by a person who did not participate in the follow-up studies. Patients with primary gastric cancer who participated in this study were recruited consecutively from February 2012 to August 2013. The study material consisted of 48 tumor tissue samples, paired paracancerous histological normal tissues (PCHNTs) which are obtained during curative surgery, and the patients’ whole blood samples preoperatively. In the meantime, another 25 blood samples from healthy individuals were also obtained. None of the experimental subjects had received prior gastric resection or preoperative chemotherapy/radiation therapy. All samples were immediately frozen and stored at −80°C until DNA was extracted. In order to reduce bias, samples were randomly coded before processing. All patients voluntarily joined this study with written informed consents to have their biologic specimens analyzed. This study was announced by the Ethical Committee of the First Affiliated Hospital of Xi’an Jiaotong University.

2.2. DNA Isolation

DNA was extracted from tissues ( mg) with the TIANamp Genomic DNA Kit and for serum samples (400 μL) by use of TIANamp Blood DNA Kit (Tiangen, China). All procedures were strictly carried out according to the manufacturer’s instructions. The concentration of DNA was measured by ultraviolet spectrophotography and the quality of DNA was tested by PCR amplification of the human β-actin.

2.3. Bisulfite Modification

As to bisulfite genomic DNA modification, 2000 Ng of DNA was modified by EpiTect Bisulfite Kit (Qiagen) to convert all unmethylated cytosine to uracil. The bisulfite-treated DNA was eluted in 20 μL of TE buffer and stored at −20°C until being processed.

2.4. Methylation-Specific Polymerase Chain Reaction (MSP)

After the bisulfite treatment, we used methylation-specific PCR to testify the methylation status of the OSR2, PPFIA3, and VAV3 promoter. The primers specific to methylated and unmethylated sequences and annealing temperature are summarized in Table 1. We repeated each experiment at least three times in order to reduce false results.
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