Promoter Methylation And The Expression Of RKIP Gene In Gastric Cardiac Adenocarcinoma

Objective: Gastric cardia adenocarcinoma (GCA) is an especial type of esophagogastric junction cancer which was one of the frequent kinds of digestive system malignant tumour in our country, especially in the north. Epidemiology shows that there was an increasing tendency between morbidity and mortality in high generating region. It has the similarly epidemiological characteristic with esophageal carcinoma in Clinical symptoms and geographical distribution. The GCA has not to readily discover in pristine period, the case of Clinical doctors'office visiting has been in medium-advanced stage and the therapeutic effectiveness is bad. It shows that the research about nosogenesis and early diagnosis of GCA is important. The molecular mechanism of generation and development in GCA has not cleared at present, it universally presumes that environmental and hereditary factor collectively induce the GCA. To decrease the morbidity and mortality of GCA, it needs to research the nosogenesis indispensably.Raf kinase inhibitory protein (RKIP; also known as PEBP, for phosphatidylethanolamine-binding protein) is an endogenous inhibitor of the Raf-Mitogen-activated protein kniase kinase (MEK)-MAP kinase pathway, which is a widely expressed protein in a variety of different species. It plays a pivotal modulatory role in several intracellular signaling cascades. It considered that the lower expression of RKIP has closely correlation with the generation, development and metastasis. In addition to the traditionary genetic mechanism, which the research about RKIP gene in abnormal expression mechanism, the methylation of the RKIP promoter which acts at the representative in epigenetic mechanism has been a hot spot about the nosogenesis of tumor. It is distinct that has CpG methylation status at the RKIP promoter had the intimate correlation in gastric cancer, colorectal cancer. The researches about the abnormal expression in GCA have not been reported in the domestic and abroad. The investigating which the methylation of the RKIP promoter plays important action in the nosogenesis of GCA will provide the evidence in molecular biology.The research, which the object comes from the region with high generating esophageal carcinoma in Hebei province, was to investigate the methylation of gene RKIP in GCA. A modified methylation specific PCR (MSP) was used to examine the methylation status in the 5'CpG island of RKIP and Reverse transcription PCR (RT-PCR) and Immunohistochemical SP methods were performed to detect RKIP mRNA and protein expression in tumors and corresponding adjacent non-cancerous tissues, and we tried to explore the relationship between gene methylation and the carcinogenesis, infiltration, metastasis and pathological differentiation of GCA, and provide a new theory and experiment evidence for pathogenesis, gene therapy and immunotherapy, clinical prognosis of gastric cardia carcinoma.Methods:1 We used a methylation specific PCR (MSP) method to examine the methylation status of the 5'CpG island of RKIP promoter in 80 cases of GCA and 51cases of corresponding adjacent non-cancerous tissues.2 We used reverse transcriptase PCR (RT-PCR) method to detect the mRNA level of RKIP gene in 28cases of GCA and 24 cases of corresponding adjacent non-cancerous tissues.3 We used Immunohistochemical SP methods (IHC) to examine the protein expression of RKIP gene in 32 cases of GCA and 32cases of corresponding adjacent non-cancerous tissues.4 SPSS 13.0 was applied to analyze the results of experiment.Results:1 RKIP gene was methylated in 52 of 80(65%) tumor specimens, which was significantly higher than that in corresponding adjacent non-cancerous tissues 17 of 51(33.3%,P<0.001).In GCA, Methylation frequencies of stageⅢandⅣtumor tissues 31 of 45(68.9%)were higher than those of stagesⅠandⅡtumor tissues 21 of 35(60%), and methylation frequencies of moderate and poor-moderate differentiation groups 35 of 52(67.3%)were higher than those in poor differentiation group 17 of 28 (60.7%), but both of them did not show significant difference(P>0.05).It shows that RKIP gene has non significant difference with the clinical data likes age, gender clinical stage, differential degree, lymph node metastasis (P>0.05).2 Positive immunoassaying of RKIP in tumor tissues (43.8%,14/32) was significantly lower than that in adjacent non-cancerous tissues(75%,24/32, P<0.05).Positive immunostaining of stageⅢandⅣtumor tissues (46.2%, 12/26)was higher than that of stagesⅠandⅡtumor tissues(33.3%,2/6),the positive immunostaining of poor differentiation group(38.5%,5/13) was also lower than those in moderate and poor-moderate differentiation groups(47.4%, 9/19). It shows that RKIP gene has non significant difference with the clinical data likes age, gender clinical stage, differential degree, lymph node metastasis (P>0.05).3 The mRNA level of RKIP in 28cases of GCA specimens was(0.67±0.41), which was significantly low than that the mRNA level of RKIP in 24cases of corresponding adjacent non-cancerous tissues (3.46±1.76,P<0.05). It shows that the protein expression RKIP gene has non significant difference with the clinical data likes age, gender clinical stage, differential degree, lymph node metastasis (P>0.05).4 The relationships between the methylation status in the 5'CpG island of RKIP and the expression of mRNA: the expression of mRNA in the positive methylation status in GCA tissue was(0.73±0.43), which was higher when compared with the negative methylation status (0.42±0.35), but the consequence did not show significant difference(P>0.05).5 The relationships between the methylation status in the 5'CpG island of RKIP and the protein expression: the protein expression in the positive methylation status in GCA tissue was(47.06%, 8∕17), the protein expression in the positive methylation status in GCA tissue was (57.14%, 4∕7), but the consequence did not show significant difference(P>0.05).6 The relationships between the protein expression and the expression of mRNA: the expression of mRNA in the positive immunoassaying of RKIP status in GCA tissue was(0.65±0.47), which was higher when compared with the negative immunoassaying of RKIP status (0.65±0.43), but the consequence did not show significant difference(P>0.05).Conclusions:1 RKIP gene was methylated in tumor specimens was significantly higher than that in corresponding adjacent non-cancerous tissues. It shows that RKIP gene has non significant difference with the clinical data likes age, gender clinical stage, differential degree, lymph node metastasis (P>0.05). It indicates that CpG methylation status at the RKIP promoter had may be related with oncogenesis of GCA, but the hypermethylation status of RKIP gene maybe have no effect in the development of GCA. The protein expression and the expression of mRNA in GCA was significantly lower than that in corresponding adjacent non-cancerous tissues, it indicates that the abnormal expression of RKIP had may be related with oncogenesis of GCA.2 The protein expression of RKIP which in the positive methylation status and the negative methylation status in GCA tissue has non significant difference, and it has non significant difference with the clinical data likes age, gender clinical stage, differential degree, lymph node metastasis. It indicates that CpG methylation status at the RKIP promoter had may be not the capital reason of the abnormal protein expression of RKIP in GCA.3 The expression of mRNA in RKIP which in the positive methylation status and the negative methylation status in GCA tissue has non significant difference, and it has non significant difference with the clinical data likes age, gender clinical stage, differential degree, lymph node metastasis. It indicates that CpG methylation status at the RKIP promoter had may be not the capital reason of the abnormal expression of mRNA of RKIP in GCA.