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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 597-600

Correlations between gastric cancer family history and ROBO2 and RASSF2A gene methylations


1 Department of Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
2 Department of Pathology, Linzhou Center Hospital, Linzhou, China

Date of Web Publication25-Jul-2016

Correspondence Address:
Zhi-Wei Chang
The First Affiliated Hospital of Zhengzhou University, Constructive East Road, Zhengzhou City
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.146089

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 > Abstract 


Objective: To explore the correlation between ROBO2 and RASSF2A gene methylations and gastric cancer family history.
Materials and Methods: ROBO2 and RASSF2A gene methylations in gastric cancer tissues and peri.cancerous tissues were detected with methylation.specific PCR in 36. patients with gastric cancer family history and 33 without gastric cancer family history. The correlations of ROBO2 and RASSF2A gene methylations with family history, and clinical and pathological characteristics were analyzed.
Results: ROBO2 and RASSF2A gene methylations were all significantly higher in gastric cancer tissues (30% and 26%) than in peri-cancerous tissues (0% and 0%) (all P < 0.05). ROBO2 gene methylation was significantly lower in the patients with gastric cancer family history (17%, 6/36) than in the patients without gastric cancer family history (41%, 15/33) (P < 0.05).
Conclusion: ROBO2 and RASSF2A gene methylations may be related to gastric tumorigenesis, and ROBO2 gene methylation is associated with sporadic gastric cancer.

Keywords: Gastric cancer, methylation, family history, RPBO2, RASSF2A


How to cite this article:
Chang ZW, Dong L, Qin YR, Song M, Guo HY, Zhu QL. Correlations between gastric cancer family history and ROBO2 and RASSF2A gene methylations. J Can Res Ther 2016;12:597-600

How to cite this URL:
Chang ZW, Dong L, Qin YR, Song M, Guo HY, Zhu QL. Correlations between gastric cancer family history and ROBO2 and RASSF2A gene methylations. J Can Res Ther [serial online] 2016 [cited 2019 Dec 16];12:597-600. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/597/146089




 > Introduction Top


Gastric cancer is one of the most common malignant tumors, and its mortality is ranked second in malignant tumors.[1] Most gastric cancer is sporadic, and its familial aggregation ranks second only to colorectal cancer and breast cancer.[2] About 10% gastric cancer show the tendency of familial aggregation, and the gastric cancer risk in the population with gastric cancer family history is 1.5-3.5 times the risk of gastric cancer in the population without gastric cancer family history.[3] However, the gastric carcinogenesis mechanism, especially the molecular phenotype differences between patients with various family histories are still not completely clear. ROBO2 is located on chromosome 3p12.3 and RASSF2A on chromosome 20p13. They may be potential tumor suppressor genes and their methylations are common in a variety of tumors.[4],[5] In this study, we detected ROBO2 and RASSF2A gene methylations in gastric cancer tissues and peri-cancerous tissues with methylation-specific PCR in order to analyze the correlations of ROBO2 and RASSF2A gene methylations with family history, and clinical and pathological characteristics.


 > Materials and Methods Top


All study methods were approved by the Ethics Committee of zhengzhou university. All the subjects enrolled into the study gave written formal consent to participate.

Subjects

From June 20, 2011 to June 20, 2012, 69 patients with gastric cancer from Zhengzhou University Hospital and Linzhou certer Hospital Hospital were enrolled in this study. Of the 69 patients, 48 were men and 21 were women, with a mean age of 58 ± 6 years (range: 43-69). The 69 patients included 36 patients with gastric cancer family history (27 men and 9 women with a mean age of 56z ± 5 years, range: 38-65) and 33 patients without gastric cancer family history (21 men and 12 women with a mean age of 58 ± 7, range: 45-69). During operation, gastric cancer tissues and peri-cancerous tissues (more than 2 cm away from gastric cancer tissues) were collected followed by immediately being put in liquid nitrogen, and then stored at -80°C for future use. All patients did not receive both radiotherapy and chemotherapy, and were diagnosed with gastric cancer by pathological examination. Gastric cancer family history positive must be in line with one of the following items: (1) Two or more than 2 patients with gastric cancer in the first- and second-degree relatives, and one patient younger than 50 years; (2) three or more than 3 patients with gastric cancer in the first- and second-degree relatives.[6] The family histories of the patients whose first- and second-degree relatives have no cancer are regarded as negative.

DNA extraction from tissues

DNA was extracted from tissues with conventional phenol/chloroform/isopropanol method

Sulfite modification

A total of 3.3 μl of 3M NaOH was added into 30 μl of DNA at 37°C for 15 min, 333 μl of sulfite was added at 55°C for 4 h, 1 ml of QX I (Qiagen) and 40 μl of QX II (16 μl/2 μg DNA) were added at room temperature with rotation for one hour, and then washed with PE. A total of 5.56 μl of 3M NaOH was added at 37°C for 15 min, 27.78 μl of 9M NH4 OAc (pH 7.0) was added with mixing, 50 μl of 3M NaOAc (pH 5.2) was added to make pH less than 7.0, 1 ml of QX I (Qiagen) was added at room temperature with rotation for 20 min, and then washed with PE after removal of supernatant.

Methylation-specific PCR

Primers were synthesized by Shanghai Sangon [Table 1]. Each sample was reacted with both methylated and unmethylated primers with gradient PCR instrument (Biometra, US). PCR products underwent 1% agarose gel electrophoresis, and results were observed with UV gel imaging system. If there was a methylated band in the sample, the sample was regarded as methylation (M). If there was an unmethylated band and there was no methylated band in the sample, the sample was regarded as unmethylation (U). SssI methyl transferase enzyme-treated human genomic DNA (NEB Company, USA) was served as positive control, untreated normal human peripheral blood DNA as negative control and distilled water as blank control.[7]
Table 1: Primers and reaction systems

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Statistical analysis

Statistical treatment was performed with SPSS17.0 software. χ2 test and Fifer's exact test were used in the comparison between groups. Statistical significance was established at P < 0.05.


 > Results Top


ROBO2 and RASSF2A gene methylations in gastric cancer tissues and peri-cancerous tissues

ROBO2 gene methylation was significantly higher in gastric cancer tissues (30%, 21/69) than in peri-cancerous tissues (0, 0/69) (P < 0.05), and RASSF2A gene methylation was also significantly higher in gastric cancer tissues (26%, 18/69) than in peri-cancerous tissues (0%, 0/69) (P < 0.05) [Table 2], [Figure 1] and [Figure 2].
Table 2: ROBO2 and RASSF2A gene methylations in gastric cancer tissues and peri-gastric cancer tissues

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Figure 1: Electrophotogram of ROBO2 gene methylation M: Methylation; U: Unmethylation; N1 and N2: Peri-cancerous tissue; T1, T2 and T3: gastric cancer tissue

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Figure 2: Electrophotogram of RASSF2A gene methylation M: Methylation; U: Unmethylation; N1 and N2: Peri-cancerous tissue; T1, T2 and T3: gastric cancer tissue

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Correlations between ROBO2 and RASSF2A gene methylations and gastric cancer family history

ROBO2 gene methylation was significantly lower in the patients with gastric cancer family history (17%, 6/36) than in the patients without gastric cancer family history (41%, 15/33) (P < 0.05). Although RASSF2A gene methylation was higher in the patients with gastric cancer family history (33%, 12/36) than in the patients without gastric cancer family history (18%, 6/33), there was no statistical difference in RASSF2A gene methylation between the two groups (P < 0.05) [Table 3].
Table 3: ROBO2 and RASSF2A gene methylations in the patients with gastric cancer family history and the patients without gastric cancer family history

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Correlations of ROBO2 and RASSF2A gene methylations with sex, differentiation and metastasis

ROBO2 gene methylation was lower in men (25%, 12/48) than in women (43%, 9/21) (P > 0.05), but RASSF2A gene methylation was higher in men (31%, 15/48) than in women (14%, 3/21) (P > 0.05). ROBO2 gene methylation was higher in well-differentiated gastric cancer (50%, 6/12) than in moderately differentiated (25%, 9/36) and poorly differentiated gastric cancer (29%, 6/21), respectively (all P > 0.05). RASSF2A gene methylation was higher in moderately differentiated gastric cancer (33%, 12/36) than in well-differentiated (25%, 3/12) and poorly differentiated gastric cancer (14%, 3/21), respectively (all P > 0.05). Both ROBO and RASSF2A gene methylations were lower in metastatic gastric cancer than in non-metastatic gastric cancer (all P > 0.05) [Table 4].
Table 4: Correlations of ROBO2 and RASSF2A gene methylations with sex, differentiation and metastasis

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 > Discussion Top


DNA methylation is an important modification, which exerts considerable influence on transcription inhibition, chromosome structural adjustment, X-chromosome inactivation and genomic imprint.[8],[9] Epigenetic characteristics caused by genomic DNA hypermethylation are common in tumor tissues. ROBO2 on human chromosome 3p12.3 is a frequent missing focus in head and neck neoplasm, and several breast cancer cell lines. Heterozygous and homozygous losses in ROBO2 have been found in lung cancer and other malignant tumors,[4],[10] and deletions occurring in ROBO2 reach 66% in prostatic cancer.[11] These suggest that ROBO2 gene is associated with tumorigenesis and is a potential tumor suppressor gene. However, little research has been done about the correlations of ROBO2 gene with gastric tumorigenesis and the genetic mechanism of gastric cancer. It is reported that there are 6% of ROBO2 gene mutations which all contain ROBO2 expression loss in gastric cancer.[12] This study found that there was ROBO2 gene methylation in gastric cancer and ROBO2 gene methylation was significantly higher in gastric cancer tissues than in peri-cancerous tissues for the first time, suggesting that ROBO2 gene may be a tumor suppressor gene and plays an important role in gastric tumorigenesis.

RASSE2 is located on human chromosome 20p13 and identified as tumor suppressor gene.[9] RASSE2 gene can promote apoptosis and cell cycle arrest. It is reported that RASSF2A gene methylation is common in colon cancer and adenoma, but it is not found in normal colonic mucosa.[13],[14] In addition, RASSF2A gene methylation is also found in gastric cancer, breast cancer, oral squamous cell carcinoma, nasopharyngeal carcinoma and endometrial cancer,[4],[15],[16],[17] suggesting that RASSF2A gene methylation may be associated with KPAS, BRAF and PIK3CA mutations in tumors.[5] RASSF2A gene methylation occurred in 100% of pancreatic cancer cell lines (8/8) and 20% of pancreatic cancer tissues, but it was not found in corresponding normal tissues.[18] This study indicated that RASSF2A gene methylation was significantly increased in gastric cancer tissues, but it was not found in peri-cancerous tissues, suggesting that RASSF2A gene methylation is frequent in gastric cancer and may plays an important role in gastric tumorigenesis.

Gene promoter methylation is frequent in sporadic and hereditary gastric cancer, and usually exists with heterozygous loss or gene mutation.[19] CDH1 promoter methylation is found in more than 50% of familial gastric cancer and 40-80% of sporadic gastric cancer.[20] However, there have not been the reports about the correlations of ROBO2 and RASSF2A gene methylations with gastric cancer family history. Our study indicated that ROBO2 gene methylation was significantly lower in the patients with gastric cancer family history than in the patients without gastric cancer family history, suggesting that low expression of ROBO2 protein caused by ROBO2 gene methylation may be associated with sporadic gastric tumorigenesis. However, how ROBO2 gene methylation is associated with sporadic gastric tumorigenesis remains to be further investigated. Our study also indicated that although RASSF2A gene methylation was higher in the patients with gastric cancer family history than in the patients without gastric cancer family history, there was no statistical significance between the two groups, suggesting RASSF2A gene methylation perhaps plays an important role in hereditary gastric tumorigenesis. It is reported that methylation frequency is related to age in the patients aged over 42 years.[21] Our study indicated that there were no statistical differences in ROBO2 and RASSF2A gene methylations between men and women; well-differentiated, moderately differentiated and poorly differentiated; and metastatic and non-metastatic.

In summary, ROBO2 gene methylation is associated with sporadic gastric tumorigenesis. This is important for further exploring gastric cancer pathogenesis.


 > Acknowledgment Top


This study was supported by the National Natural Science Fundation of China (NO. 81150010 and NO. 81272227)

 
 > References Top

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Hesson LB, Wilson R, Morton D, Adams C, Walker M, Maher ER, et al. CpG island promoter hypermethylation of a novel Ras-effector gene RASSF2A is an early event in colon carcinogenesis and correlates inversely with K-ras mutations. Oncogene 2005;24:3987-94.  Back to cited text no. 14
    
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Park HW, Kang HC, Kim IJ, Jang SG, Kim K, Yoon HJ, et al. Correlation between hypermethylation of the RASSF2A promoter and K-ras/BRAF mutations in microsatellite-stable colorectal cancers, Int J Cancer 2007;120:7-12.  Back to cited text no. 15
    
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Maruyama R, Akino K, Toyota M, Suzuki H, Imai T, Ohe-Toyota M, et al. Cytoplasmic RASSF2A is a proapoptotic mediator whose expression is epigenetically silenced in gastric cancer, Carcinogenesis 2008;29:1312-8.  Back to cited text no. 16
    
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Zhang Z, Sun D, Van do N, Tang A, Hu L, Huang G. Inactivation of RASSF2A by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinoma, Int J Cancer 2007;120:32-8.  Back to cited text no. 17
    
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Zhao L, Cui Q, Lu Z, Chen J. Aberrant methylation of RASSF2A in human pancreatic ductal adenocarcinoma and its relation to clinicopathologic feature, Pancreas 2012;41:206-11.  Back to cited text no. 18
    
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Machado JC, Oliveira C, Carvalho R, Soares P, Berx G, Caldas C, et al. E-cadherin gene (CDH1) promoter methylation as the second hit in sporadic diffuse gastric carcinoma. Oncogene 2001;20:1525-8.  Back to cited text no. 19
    
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Zazula M, Ferreira AM, Czopek JP, Kolodziejczyk P, Sinczak-Kuta A, Klimkowska A, et al. CDH1 gene promoter hypermethylation in gastric cancer: Relationship to Goseki grading, microsatellite instability status, and EBV invasion. Diagn Mol Pathol 2006;15:24-9.  Back to cited text no. 20
    
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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