| Objectives:Esophageal cancer is more common in the world. Its morbidity is about 69-166 /100 thousand. More than 300 thousands new patients increased every year. And most of them are in the developing country. It is estimated that about 200 thousand people died of it each year all over the world. At present, its incidence ranked the sixth of the most commom malignant tumor in the world, but it ranked fourth in China. It is one of the least studied and deadliest cancers, with a remarkable geographical distribution and a low likelihood of cure. The tumorigenesis is closely related to genetics, race, living habits, eating habits and oral hygiene. Its development is a complex process by accumulation of the multi-genes mutation in which many oncogenes and tumor suppressor genes are involved, but its exact pathogenesis is not clear. At present, the diagnosis of esophageal cancer mainly depends on barium meal and esophagoscopy detection, many patients are already at the advanced stage at the time of diagnosis. There is lack of an effective method of early diagnosis clinically. Therefore, the current challenges in the management of it are to obtain a better understanding of the underlying molecular biological alterations to provide an effective treatment options.Over the last decade, Barrett' esophagus is to a clear upward trend in our country. Because of the esophagogastric reflux, the normal squamous epithelium is replaced by the columnar epithelium which has a strong ability to resist digestive juice. It is often with the peptic ulcer, esophageal stricture, intestinal metaplasia, dysplasia and up to esophageal adenocarcinoma. It is considered that the risk of Barrett's esophagus turn to the esophageal cancer is 30 ~ 125 times higher than that of the normal people. About 50% patients with severe dysplasia of Barrett's esophagus may develop to esophageal adenocarcinoma. The evolution process of the Barrett's esophagus turn to the adenocarcinoma includes three steps, metaplasia - heteromorphosis - adenocarcinoma. Therefore it is considered as a precancerous lesion of esophageal cancer. It is very necessary to strengthen the monitoring of patients with Barrett's esophagus.Besides the genetics, such as point mutation, insertion, deletion, ectopia, epigenetics is another important mechanism of gene inactivation, it does not change the gene sequences and can be inherited and can lead to malignant tumor-related genes (including proto-oncogenes and tumor suppressor genes) dysfunction. Includes methylation/demethylation, histone acetylation/deacetylation status. DNA methylation is one of the earliest epigenetic modified ways and certified to be associated with multiple tumorigenesis. Study shows that DNA methylation can change chromatin structure, DNA conformation, DNA stability and DNA-protein interaction therefore can control gene expression.DNA methylation refers to the process that catalysised by DNA methyltransferase (DNMTs), consider the S-adenosyl-L-methionine (SAM) as a methyl donor and transfer it to the 5mC of cytosine. In mammals there are at least three DNMTs involved in the formation and maintenance of DNA methylation. Included DNMT1,DNMT3a and DNMT3b. There are two main forms, de novo methylation and maintenance methylation. DNMTs activity can increase the occurrence of abnormal DNA methylation and chromosome instability. The abnormal expression of DNMTs may influence multiple genes, including SLC5A8 and CpG island methylation phenotype.In tumor tissues there is a abnormal phenomenon that the whole genome of a wide range of low-methylation, and local of it of the hypermethylation. It is an important feature and also important reason of tumor occurrence and development. DNA methylation reduces the binding affinity of gene sequences and transcription factors. Interference the transcription initiation and make the gene silenced. This phenotype has not changed the gene sequences, but it plays an important role in regulating the expression of tumor-related genes. Mutiple genes and sites of DNA methylation at the same time also known as CpG island methylation phenotype (CIMP). The prognosis and mechanism of occurrence and development of the tumor with different CIMP are different. Many researches have shown that CIMP involves the regulation of gene expression of a series of tumor-associated genes, and it plays an important role in the occurrence and development of tumors. At present, the studying of the aberrant methylation status of tumor-associated genes is hot spot of the occurrence mechanism of tumors.In recent years, the studies found that tumor suppressor gene SLC5A8 (the solute carrier family 5 member 8 gene)is closely related to the occurrence of variety human tumor. SLC5A8 is located in human chromosome 12q22-23, is an NA / glucose co-transporter protein family members, and is a carrier coupled by the NA transporter of short chain fatty acids and other single-carboxylic acid, such as lactate, pyruvate. At the same time, it is also a tumor suppressor gene, its exon 1 of the CpG island methylation and histone acetylation can make the gene expression silenced and promote the occurrence of tumors. In a variety of colon tumor markers, it is the first membrane transporter protein associated with the tumor suppressor function. But the status of SLC5A8 methylation is unknown in Barrett esophagus and esophageal cancer.This topic will study the clinical specimens of esophageal cancer tissues postoperatively, and its precancerous lesions-Barrett esophagus and postoperative and preoperative plasma of the esophageal cancer patients in Dalian, and adjacent tissues and normal mucosa as a control.The methylation specific-PCR (MSP) was used to detect the tumor suppressor gene SLC5A8 methylation in the above-mentioned tissues. And the RT-PCR and Western-blot were used to detect the gene expression from the mRNA and protein level in esophageal carcinoma, adjacent tissues and normal mucosa. To explore the role of it in the occurrence and development of esophageal cancer, and the relationship associated with the clinical relevant factors. And further analysis of the relationship between the methyl transferase and promoter region of SLC5A8 methylation status, and the characteristics of methylation of the genome-wide in esophageal cancer tissues. Dalian area is with a high incidence of esophageal cancer. The studies of the molecular changes of the occurrence and development of esophageal cancer, can help people to insight into the mechanism of the occurrence of esophageal cancer happened in the region. It is very important of it to enact the preventative measures, look for the early diagnosis methods and treatment strategies and the judgement of prognosis.Methods:Forty-five surgical esophageal cancer specimens, include cancerous tissues, adjacent tissues and normal mucosa (38 male, 7 female, mean age 60 years), 30 cases of preoperative and postoperative plasma and 42 cases of Barrett's esophagus specimens were selected from the first affiliated hospital of Dalian Medical University. All of the patients were long-term Dalian residents who never received pre-operative chemotherapy.By the methods of MSP to detect the methylation status of different tissues, RT-PCR and Western blotting were used to detect the expression status of SLC5A8. And further analysis of the relationship between the methyl transferase and promoter region of SLC5A8 methylation status, and the characteristics of methylation of the genome-wide in esophageal cancer tissues.The data were statistically analyzed byχ2 test and Fisher exact probability method with SPSS 12.0 software.Results:1. The expression status of SLC5A8 gene in esophageal cancer tissues, adjacent tissues and cutting edge tissues.(1). RT-PCR. The expression status of SLC5A8 gene in esophageal cancer tissues, adjacent tissues and cutting edge tissues were 26.7% (12/45), 73.3% (33/45) and 91.1% (41/45), respectively. The rate of gene expression in cancer tissues was significantly lower than that in adjacent tissues and cutting edge tissues (χ2 = 19.6, P<0.01;χ2 = 38.59, P<0.01).(2). In order to verify the results of RT-PCR, the Western-blot was used to detect the protein expression of SLC5A8 in esophageal cancer tissues, adjacent tissues and cutting edge tissues. The results are similar to that of the RT-PCR. They are 25.0%(2/8), 75.0%(6/8), 87.5%(7/8), respectively.2. The methylation status of SLC5A8 in esophageal cancer tissues, adjacent tissues and cutting edge tissues.(1). The methylation-positive rate of SLC5A8 in 45 cases of esophageal cancer tissues, adjacent tissues and cutting edge tissues were 68.8% (31/45), 11.1% (5/45), 4.4% (2/45), respectively. The frequency of methylation in tumor tissue was remarkably higher than that in adjacent tissue and cutting edge tissue(χ2=31.3, P<0.01).(2). There was not significant difference between adjacent tissues (11.1%) and cutting edge tissues (4.4%) (χ2 = 1.39, P>0.05). There were no correlations among the methylation status of SLC5A8 in esophageal cancer tissue and patient's sex, age, tumor location, degree of differentiation, TNM staging and the pathological type (P>0.05).3. The relationship between the gene expression status and methylation status of SLC5A8 in esophageal cancer tissues.(1). The gene expression rate of SLC5A8 in 45 esophageal carcinoma tissues was 26.7% (12/45), the gene expression rate in cancer tissues was significantly lower than that of adjacent tissues and cutting edge tissues.(2). Only 2 cases were detected the expression of SLC5A8 gene in 31 cases of methylation-positive tumor tissues and 10 cases were detected the expression in 14 cases of methylation-negative tumor tissues. There was statistical significance between them(χ2 =21.168,P<0.001).4. The methylation status of SLC5A8 in Barrett's esophagus.The methylation status of SLC5A8 in Barrett's esophagus is 35.7% (15/42). The methylation-positive rate (35.7%) of SLC5A8 in Barrett's esophagus was significantly higher than that of its paired normal esophageal mucosa (9.5%). The difference was statistically significant (χ2 = 8.23, P <0.01). The methylation status of SLC5A8 gene with the progress of different pathological (normal mucosa- Barrett's esophagus - esophageal cancer) showed an increasing trend, there were significant differences among the different pathological stages (χ2 = 8.23, P<0.01.χ2 = 9.59, P <0.01).5. The methylation status of SLC5A8 in preoperative and postoperative plasma of esophageal cancer patients.The methylation rate of SLC5A8 gene in preoperative and postoperative plasma of 30 cases of esophageal cancers were 43.3% (13/30), 16.7% (5/30), respectively. 8 of 13 esophageal cancer patients with SLC5A8 gene methylation-positive in preoperative plasma turned negative after their radical surgery, the rate of methylation of postoperative plasma was significantly lower than that of preoperative plasma (χ2 = 5.08, P<0.05). There were not SLC5A8 methylation in preoperative and postoperative plasma of the esophageal carcinoma patients with the methylation-negative tissues.6. The correlation between the expression of methyltransferase and the promoter region of SLC5A8 methylation status.(1).The expression rates of DNMT1, DNMT3a, DNMT3b in 45 cases of esophageal cancer patients with tumor tissue and normal mucosal tissues were 64.4% (29/45), 22.2% (10/45), 24.4% (11/45), 31.1% (14/45) and 31.1% (14/45), 33.3% (15/45), respectively. The difference of the DNMT1 expression between the tumor tissues and normal mucosal tissues was statistically significant (χ2=16.33, P<0.005).(2).The difference expression among the DNMT1 and DNMT3a, DNMT3b in the methylation-positive tumor tissues (31 cases) were statistically significant (χ2=6.97, P<0.05).7. Whole-genome methylation status of esophageal carcinoma.(1) There was lower methylation tendency in the whole- genome DNA of esophageal carcinoma tissues.(2)There was no significant difference of methylation status between the cancerous tissues and paired adjacent noncancerous tissues in the same patients.Conclusions:1. The expression of SLC5A8 was lower in esophageal cancer, methylation is one of the important factors of its low expression. Methylation can make it silenced and decrease its expression, influence its normal physiologic function, and induce the occurrence and development of cancer.2. Methylation phenomenon of the SLC5A8 can occur during the esophageal precancerous lesions- Barrett's esophagus, and with the increased of the pathological progress, methylation status showed an obvious upward trend from the normal mucosa, Barrett esophagus to esophageal cancer tissue. Suggesting that tumor suppressor gene SLC5A8 methylation is an early molecular event of esophageal cancer and it is one of the mechanisms for Barrett's esophagus transformed into esophageal cancer.3. The detection of SLC5A8 gene methylation in plasma could be used as a convenient and atraumatic method. It could be used in the field of monitoring the reaction of radiotherapy and hemotherapy, judging the prognosis if combined detection of methylation of other multiple TSGs.4. The expression of DNMT1 was high in esophageal cancer with the hypermethylation of promoter region of SLC5A8, DNMT1 participate in maintenance methylation. But the expression of DNMT3a and DNMT3b was not related to the hypermethylation of promoter region of SLC5A8, there participate in the de novo methylation. Regulation to the expression of DNMT1 could change the CIMP of esophageal cancer in this region.5. There was lower methylation tendency in the whole- genome DNA of esophageal carcinoma tissues.6.SLC5A8 gene may become an important index of early diagnosis of esophageal cancer, and the new target of the gene therapy.In summary, we detected the status of tumor suppressor gene SLC5A8 methylation and expression status in esophageal cancer and Barrett's esophagus, and the changes of methylation status of preoperative and postoperative plasma, the relationship between methyltransferases and methylation status of SLC5A8 gene, and the characteristic of methylation status of the genome-wide. Analyzed the relationship between the methylation of promoter region of SLC5A8 and the occurrence and development of esophageal cancer by epigenetic. It can help us to reveal the epigenetic characteristic of the occurrence and development of esophageal cancer. It provided the theory bases to use epigenetic into the early diagnosis, monitoring the effect of treatment and judgment of prognosis of esophageal cancers.
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