Detection Of Serum RASSF1A Gene Promoter Hypermethylation In Patients With Gastric Cancer

【Background】Gastric cancer is a worldwide incidence of second tumors,it is estimated that one-tenth of all tumors,the prognosis is poor.In China,the mortality rate of gastric cancer is the first resident of the tumor.Upper gastrointestinal endoscopy is now recognized as the most effective method of gastric cancer,but as a means of population census acceptance rate than the arrival,but also for small gastric cancer have a certain rate of missed diagnosis.Ⅰ～Ⅲgastric cancer patients in general surgery,but patients with advanced gastric cancer surgery has lost the opportunity to seriously affect the prognosis.Therefore,to explore new diagnostic methods to improve the rate of early diagnosis of gastric cancer is essential.Unfortunately,the diagnosis of gastric cancer has been the relative lack of sensitivity and specificity of tumor markers.The search for new molecular markers is to increase the rate of early diagnosis of gastric cancer an important research direction.Research in recent years prompted many of the promoter region of tumor suppressor gene CpG island aberrant methylation of tumor detection may be molecular markers for early diagnosis.Recent studies have shown that the new candidate tumor suppressor gene family of Ras-related region 1A(Ras association domain family 1 A,RASSF1A) promoter region of CpG island aberrant methylation of tumor suppressor gene inactivation is the main mechanism for the occurrence of gastric cancer,the development of plays an important role.Recent studies have shown that gastric cancer patients in the tumor tissue,blood can be detected in a higher frequency of RASSF1A gene promoter methylation abnormalities,and the frequency of methylation in patients with gastric cancer between clinicopathological parameters associated with a certain sex,suggesting that RASSF1A promoter methylation abnormalities in gastric cancer with a certain degree of clinical significance.With the emergence of circulating nucleic acid studies have confirmed that serum tumor suppressor gene methylation in clinical research with the prospect of some.At present,gastric cancer patients with RASSF1A promoter methylation abnormality is very limited research,the domestic has not been reported.【Objective】The past,such as the CEA tumor marker for diagnosis of gastric cancer have a lot of limitations.Search for new tumor markers for early diagnosis of gastric cancer prospects.The main purpose of this study was to test the serum of patients with gastric cancer,tumor tissue and tissue adjacent the promoter region of RASSF1A gene methylation abnormalities and to explore its relationship with the relationship between clinical parameters,to explore its early diagnosis and prognosis of gastric cancer to assess the possible role.【Materials and Methods】Methylation-specific polymerase chain reaction(methylation special PCR, MSP) is a fast,sensitive detection of gene methylation,high sensitivity,can be used for trace analysis and analysis of homologous gene,the method can not only avoid the restrictive endonuclease digestion and not entirely caused by false negative and false positive rate is low and serum for the detection of gene methylation from trace possible.It will first DNA bisulfite treatment,this is not the cytosine methylation into uracil,and the methylation of the same,followed by line-specific primer PCR. MS-PCR in the design of two pairs of primers,and asked:1.End primers are designed to detect the end of site;2.Two pairs of primers,respectively,and only after bisulfite sequencing of the complementary pair,that is a combination of treatment Methylation of DNA chain,and the other on the combined treatment of non-methylated DNA chain.Detection of PCR products of MSP,if the response to treatment with DNA methylation primer chain fragment can be amplified,then detected the presence of methylated sites;if used for treatment of non-methylated DNA chain amplified a fragment,then the site has been detected methylation does not exist.In this paper,MSP detected 47 cases of gastric cancer patients,30 cases of the stomach in patients with benign lesions and 30 healthy controls the serum RASSF1A gene promoter methylation in the promoter region and to explore its relationship with the relationship between the clinical parameters.Of these,16 patients with gastric cancer at the same time dedicated surgical resection of cancer tissue,cancer tissue specimens,as well as preoperative and postoperative control study of blood samples.【Results】Gastric cancer patients with the promoter region of RASSF1A gene methylation was 34.0%(16/47),benign lesions in patients with gastric methylation was only 3.3%(1/30) and healthy controls were no methylation;gastric methylation in patients was significantly higher than the stomach in patients with benign lesions and healthy controls(P=0.0015).16 cases of gastric adenocarcinoma tissue samples in 5 cases(31.2%) detected in the promoter region of RASSF1A gene methylation,1 case(6.25%) cancer tissues also methylation;5 cases of positive samples detected in the organization of the four cases(75.0%) preoperative and postoperative serum samples were detected by methylation, methylation-positive carcinoma of the serum positive rate of methylation of 80%. Gastric cancer patients with RASSF1A gene methylation with gender,age,degree of tumor differentiation,metastasis and serum CEA level were not related to each other.Have distant metastasis rates were slightly higher than the non-methylation of transfer,the surgical group was slightly lower than the rate of methylation of non-operation group,but the difference was no statistical difference.【Conclusions】In this paper,the use of methylation-specific polymerase chain reaction(MSP) detected 47 cases of patients with gastric adenocarcinoma,30 cases of the stomach in patients with benign lesions and 30 healthy controls the serum RASSF1A gene promoter methylation in the promoter region,of which 16 cases of gastric cancer patients At the same time,organizations dedicated surgical resection of cancer, cancer tissue specimens,as well as preoperative and postoperative control study of blood samples and to study its relationship with the relationship between the clinical parameters.The results of the following fruit:1) in peripheral blood serum in patients with gastric cancer gene DNA RASSF1A promoter region CpG island of a higher frequency of abnormal methylation:gastric cancer patients with the promoter region of RASSF1A gene methylation was 34.0% (16/47),benign lesions in patients with gastric methyl rate of 3.3%(1/30) and healthy controls were no methylation;methylation in patients with gastric cancer was significantly higher than the stomach in patients with benign lesions and healthy controls(P=0.0015).2) 16 cases of gastric adenocarcinoma tissue samples in 5 cases(31.2%) detected in the promoter region of RASSF1A gene methylation,1 case(6.25%) cancer tissues also methylation;5 cases of positive samples detected in the organization of the 4 cases(75.0%) preoperative and postoperative serum samples were detected by methylation,methylation-positive carcinoma of the serum positive rate of methylation of 80%.3) gastric cancer patients with RASSF1A gene methylation and sex,age,degree of tumor differentiation,metastasis and serum CEA level were not related to each other.Have distant metastasis rates were slightly higher than the non-methylation of transfer,the surgical group was slightly lower than the rate of methylation of non-operation group,but the difference was no statistical difference. Thus serum RASSF1A gene promoter region methylation status of CpG islands is expected for the detection of gastric cancer diagnosis and prognosis to determine the means to provide a new auxiliary.