Promoter Methylation And MRNA Expression Of TβRⅠ, Ⅱ Gene In Esophageal Squamous Cell Carcinoma

Objective: Esophageal carcinoma is one of the most common digestive system malignant tumor in China, and above 95% esophageal carcinoma is esophageal squamous cell carcinoma(ESCC). China is the country where the morbility and mortality of esophageal carcinoma is the highest. Because of lacking early diagnosis biomarkers, the patients have entered the clinical medial-advanced stage, and have missed the best diagnosis and therapy period. Though some report shows that the morbility of esophageal carcinoma has descendent trend, there are also hundreds thousands of people who die of esophageal carcinoma every year. In order to decrease the morbility and mortality of esophageal carcinoma, we should research its pathogenesis, so as to offer reliable biomarkers and methods for prevention, early diagnosis and therapy.In the process of tumorigenesis, gene methylation is an important epigenetic mechanism. Some research has proved that many genes methylation possess the characteristic of esophageal squamous cell carcinoma. The purpose of this study was to investigate the promoter methylation and the mRNA level of transforming growth factorβreceptor I,II (TβRI,II)genes in ESCC and adjacent non-cancerous tissue, and investigate the relationship between it and the clinical data, and provide a new theory and experiment evidence for the role of TβRI,II genes methylation in the process of formation and development of ESCC.Methods:1.We used modified methylation specific PCR (MSP) method to examine the methylation status of the 5' CpG island of TβRI and TβRII gene in 65 cases of ESCC and 59 cases of adjacent non-cancerous tissue.2.We used reverse transcriptase PCR (RT-PCR) method to detect the mRNA level of TβRI and TβRII gene in 41 cases of ESCC and 15 cases of adjacent non-cancerous tissue.3.SPSS13.0 was applied to analyze the results of experiment.Results:1. Methylation status:(1) TβRI gene was methylated in 34 of 65(52.3%) ESCC specimens and 24 of 59(40.7%) adjacent non-cancerous tissue, but there was no significant difference between them. Methylation frequencies of TβRI gene had difference in different age and gender groups, but no significance (P>0.05). Methylation frequencies of TβRI gene in lymph node metastasis group 41.4% was lower than that in no lymph node metastasis group 61.1%, and methylation frequencies of TβRI gene in poor differentiation group 40.0% was lower than moderate and poor-moderate differentiation groups 54.5%, and methylation frequencies of TβRI gene inⅠ,Ⅱstage 56.8% was higher thanⅢ,Ⅳstage 46.4%, but all of them did not show significant difference (P>0.05).(2) TβRII gene was methylated in 46 of 65(52.3%) ESCC specimens, which was significantly higher than that in adjacent non-cancerous tissue 47.5%(28/59()P<0.05). Methylation frequencies of TβRII gene had difference in different age and gender groups, but no significance (P>0.05). Methylation frequencies of TβRII gene in lymph node metastasis group 65.5% was lower than that in no lymph node metastasis group 75.0%, and methylation frequencies of TβRII gene in poor differentiation group 60.0% was lower than moderate and poor-moderate differentiation groups 72.7%, and methylation frequencies of TβRII gene inⅠ,Ⅱstage 73.0% was higher thanⅢ,Ⅳstage 67.9%, but all of them did not show significant difference (P>0.05).(3) Methylation analysis of two genes in common: TβRI and TβRII genes methylated simultaneously in 31 cases of ESCC tissues, and in 18 cases of adjacent non-cancerous tissue. Homeochronous methylation frequencies of two genes was higher in ESCC group (47.7%) than in adjacent non-cancerous group (30.5%), but difference had no significance (P>0.05). Two genes methylation rate had no relationship with lymphatic metastasis, pathology grades and clinic stages(P >0.05).⑷The results of sequencing:methylated and unmethylated production completely fit the bill.2. mRNA expression:(1) TβRI gene mRNA expression quantity in ESCC group(0.77±0.17) was significantly lower than adjacent non-cancerous group(0.99±0.31)(P <0.05).(2) TβRII gene mRNA expression quantity in ESCC group(0.89±0.30) was significantly lower than adjacent non-cancerous group(1.10±0.29)(P <0.05).(3) Two genes mRNA expression had no linearity correlation in ESCC group(P >0.05).3.The relationship between methylation and mRNA expression: TβRI gene mRNA expression quantity was lower in methylated ESCC tissues (0.74)than unmethylated ESCC tissues(0.81), but difference had no significance (P>0.05). TβRII gene mRNA expression quantity was lower in methylated ESCC tissues (0.86) than unmethylated ESCC tissues (0.95), but difference also had no significance (P>0.05).Conclusions:1.The methylation incidence of TβRI gene had no significant difference between ESCC group and adjacent non-cancerous group, and there was no relationships between its methylation and clinical data. They indicated that the promoter methylation of TβRI gene may be not related with oncogenesis and development of ESCC. The methylation incidence of TβRII gene in ESCC group was significantly higher than that in adjacent non-cancerous group, but there was also no relationships between its methylation and clinical data, which indicated that the promoter methylation of TβRII gene may be related with oncogenesis of ESCC, but not related with development of ESCC.2.Homeochronous methylation frequencies of two genes had no significantly diference between ESCC group and adjacent non-cancerous group, and two genes methylation rate had no relationship with lymphatic metastasis, pathology grades and clinic stages. They indicated that congenerous methylation of two genes maybe had no relation to oncogenesis and development of ESCC, either.3.Expression quantity of TβRI and TβRII gene mRNA in ESCC group was significantly lower than adjacent non-cancerous group, indicating that abnormal expression of the two genes mRNA may be related with oncogenesis of ESCC.4.The expression difference of two genes mRNA was not significantly between methylated ESCC tissues and unmethylated ESCC tissues, which indicated that methylation may be not the main reason that causes the abnormal expression of mRNA.