| Objective: Esophageal cancer is one of the common malignant tumors of human and one of the highest deadly cancers. Esophageal squamous cell carcinoma is a major type of esophageal cancer. Smoking, more red meat, and lower socioeconomic status are considered to be associated with the high incidence of esophageal cancer. Although there are some progresses in the treatment of esophageal cancer, the overall survival rate of esophageal cancer patients is still not improved obviously.Epigenetic mechanisms are the connection bond of Genomics and Proteomics. DNA methylation is the most in-depth study of epigenetic regulation, and its importance is mainly reflected early diagnosis and estimation of prognosis in tumor patients with malignant tumor. DAPPER family includes three members: DACT1, DACT2 and DACT3. Human DACT1, 2, 3 genes are respectively encoded by 799, 744 and 610 amino acids protein, and located in human chromosome 14q22.3, 6q27 and 19q13.32. Sequence search respectively found 2, 1, 5 Cp G islands within DACT1, 2, 3 genes promoter region. We speculate these genes may exist methylation modify phenomenon. In the present study, we investigated the methylation status of promoter region of DACT1, 2, 3 genes in esophageal cancer cell lines, ESCC tumor tissues and the corresponding normal tissues and analyzed the relevance of the methylation status and m RNA expression of the three genes.Methods: 1 Reverse transcription polymerase chain reaction(RT-PCR) method was used to detect the m RNA levels of DACT1, 2, 3 in esophageal cancer cell lines, ESCC tumor tissues and the corresponding normal tissues. 2 Methylation specific PCR(MSP) method was used to examine methylation status of DACT1, 2, 3 genes in esophageal cancer cell lines,ESCC tumor tissues and corresponding normal tissues. 3 SPSS13.0 was applied to analyze the results of experiments.Results: 1 The m RNA expression and methylation status of DACT1, 2, 3 in esophageal cancer cell lines1.1 The m RNA expression of DACT1, 2, 3 in esophageal cancer cell lines treated or untreated with 5-aza-d CTE1, Eca109 cell lines showed weak positive expression of DACT1 while TE13, T.TN cell lines showed no expression of DACT1; TE13 cell line showed weak positive expression of DACT2 while TE1, T.TN and Eca109 cell lines showed no expression of DACT2; Eca109 cell line showed weak positive expression of DACT3 while TE1, TE13 and T.TN cell lines showed no expression of DACT3.After treatment with 5-aza-d C, DACT1 can be detected in TE13 and T.TN cell lines, and higher m RNA expression of DACT1 was detected in TE1 and Eca109 cell lines. The m RNA expression of DACT2 and DACT3 can be detected in the four treated esophageal cancer cell lines.1.2 The methylation status of DACT1, 2, 3 in esophageal cancer cell lines treated or untreated with 5-aza-d CDACT1 gene was fully methylated in TE13 and T.TN cell lines, while semi-methylation of DACT1 was detected in TE1 and Eca109 cell lines. DACT2 gene was fully methylated in TE1, T.TN and Eca109 cell lines, while semi-methylation of DACT2 was detected in TE13 cell line. DACT3 gene was detected unmethylation status in the four treated cell lines.After treatment with 5-aza-d C, the methylation level of DACT1 in TE1 cell line was decreased, the unmethylation level of DACT1 in TE1 cell line was increased, and DACT1 gene was detected unmethylation status in TE13, T.Tn and Eca109 cell lines. DACT2, 3 genes were detected unmethylation status in the four treated cell lines.2 The m RNA expression of DACT1, 2, 3 genes in ESCC2.1 The m RNA expression of DACT1 gene in ESCCDACT1 m RNA expression in corresponding normal tissues(0.91±0.77) was significantly higher than that in tumor tissues(0.60±0.48)(t=-2.413, P=0.019). The m RNA expression of DACT1 was associated with status of lymph node metastasis(P<0.05), however, it was not associated with pathological differentiation, TNM stage, age and gender of ESCC patients(P>0.05).2.2 The m RNA expression of DACT2 gene in ESCCDACT2 m RNA expression in corresponding normal tissues(0.95±0.64) was significantly higher than that in tumor tissues(0.66±0.53)(t=-2.439, P=0.018). The m RNA expression of DACT2 was correlated with status of lymph node metastasis(P<0.05), however, it was not associated with histological differentiation, TNM stage, age and gender(P>0.05).2.3 The m RNA expression of DACT3 gene in ESCCDACT3 m RNA expression in corresponding normal tissues(1.02±0.52) was significantly higher than that in tumor tissues(0.67±0.49)(t=-3.786, P=0.000). The m RNA expression of DACT3 was not associated with status of lymph node metastasis, pathological differentiation, TNM stage, age and gender of ESCC patients(P>0.05).3 The methylation status of DACT1, 2, 3 in ESCC 3.1 The methylation status of DACT1 in ESCCThe promoter methylation frequency of DACT1 in corresponding normal tissues(15.4%, 8/52) was significantly lower than that in tumor specimens(48.1%, 25/52)(P=0.000). Methylation frequency of DACT1 gene in moderate and high differentiation group(30.7%, 8/26) was much lower than that in poor differentiation group(65.3%, 17/26)(χ2 =6.240, P=0.012). Methylation frequency of DACT1 inⅠ+Ⅱ stage group(35.7%, 15/42) was much lower than that in Ⅲ + Ⅳ stage group(90.0%, 9/10)(χ2 =9.578, P=0.002). Methylation frequency of DACT1 in no lymph node metastasis group(20.0%, 4/20) was significantly lower than that in lymph node metastasis group(62.5%, 20/32)(χ2 =8.945, P=0.003). Methylation status of DACT1 gene was not associated with age and gender(P>0.05).3.2 The methylation status of DACT2 in ESCCThe promoter methylation frequency of DACT2 in corresponding normal tissues(21.1%, 11/52) was significantly lower than that in tumor specimens(50.0%, 26/52)(P=0.002). Methylation frequency of DACT2 gene in moderate and high differentiation group(34.6%, 9/26) was much lower than that in poor differentiation group(65.3%, 17/26)(χ2 =4.923, P=0.027). Methylation frequency of DACT2 in Ⅰ+Ⅱ stage group(40.4%, 17/42) was much lower than that in Ⅲ + Ⅳ stage group(90.0%, 9/10)(χ2 =7.924, P=0.005). Methylation frequency of DACT2 in no lymph node metastasis group(25.0%, 5/20) was significantly lower than that in lymph node metastasis group(65.6%, 21/32)(χ2 =8.125, P=0.004). Methylation status of DACT2 gene was not associated with age and gender(P>0.05).3.3 The methylation status of DACT3 in ESCCThe promoter methylation frequency of DACT3 in tumor specimens(11.5%, 6/52) was not significantly different with corresponding normal tissues(7.7%, 4/52)(P=0.506).4 Relationship between methylation status of DACT1, 2, 3 and its expression4.1 Relationship between methylation status of DACT1 and its expressionThe m RNA expression of DACT1 in tumor tissues with promoter hypermethylation of the gene(0.45±0.36) was significantly lower than that in tumor tissues with unmethylation of the gene(0.74±0.53)(t=-2.250, P=0.029).4.2 Relationship between methylation status of DACT2 and its expressionThe m RNA expression of DACT2 in tumor tissues with promoter hypermethylation of the gene(0.46±0.32) was significantly lower than that in tumor tissues with unmethylation of the gene(0.78±0.61)(t=-2.341, P=0.023).4.3 Relationship between methylation status of DACT3 and its expressionThe m RNA expression of DACT3 in tumor tissues with promoter hypermethylation of the gene(0.65±0.38) was not significantly different than that in tumor tissues with unmethylation of the gene(0.68±0.50)(t=-0.120, P=0.905).Conclusions:1 The expression of DACT1, 2, 3 in ESCC tumor tissues was significantly lower than that in the corresponding normal tissues, suggesting that DACT1, 2, 3 gene may be a candidate tumor suppressor gene in ESCC, and the decreased expression of them may be one of the pathogenesis of ESCC.2 There seemed to be a correlation between the low expression of DACT1, 2 and ESCC, and promoter hypermethylation of DACT1, 2 may be one of the reasons that lead to down-expression of DACT1, 2 in ESCC.3 The methylation status of DACT1, 2 was related to pathological differentiation and lymph node metastasis, indicating that promoter methylation of DACT1, 2 may be closely related to development and prognosis of ESCC.4 The methylation status of DACT3 promoter was not correlated with its expression, indicating that promoter methylation of DACT3 may be not the mechanism which leads to the inactivation of the gene. |
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