The Expression And Methylation Status Of MiR-203a Gene And MiR-203b Gene In Esophageal Squamous Cell Carcinoma

Objective: Esophageal cancer is occurred in esophageal mucosa epithelial malignant tumors, According to the histological features of esophageal cancer can be divided into five types, of which the most common esophageal squamous cell carcinoma, accounting for about 90 percent, followed by esophageal adenocarcinoma, accounting for about 7 percent, other types are rare. China is a country with high incidence and mortality regions of esophageal cancer, and the average annual incidence and mortality were 20.14/10 ten thousand and 16.7/10 ten thousand. The prognosis of esophageal cancers is poor, The 5-year survival rate is about 10% in middle or advanced stages patients. So far, the pathogenesis of esophageal cancer is still unclear.In the etiology of esophageal cancer, epigenetic changes, especially aberrant DNA methylation of the promoter region is the current research focus. In this study, the expression and methylation status of mi R-203 a and mi R-203 b were detected in esophageal cancer cell lines and esophageal squamous cell carcinoma to explore the relationship between gene methylation and development and occurrence of ESCC. Thus we provide a new theory and experimental evidence for pathogenesy, therapeutic schedule and clinical prognosis of ESCC.Methods:1 Quantitative real-time RT-PCR(q RT-PCP) method was used to detect the expression mi R-203 a and mi R-203 b gene in esophageal cancer cell lines(TE1, TE13, Yes-2, ECa109, T.TN) and the cell lines treated with 5-aza-d C, 54 ESCC tumor tissues and corresponding normal tissues.2 Methylation specific PCR(MSP)method was used to examine the methylation status of mi R-203 a and mi R-203 b gene in esophageal cancer cell lines and the cell lines treated with 5-aza-d C, 83 ESCC tumor tissues and corresponding normal tissues.3 SPSS19.0 was applied to analyze the results of experiments.Results: 1 The expression and methylation of mi R-203 a gene in ESCC 1.1 The expression and methylation status of mi R-203 a in esophageal cancer cell linesRelative low expression and hypermethylation of mi R-203 a was detected in the five esophageal cancer cell lines untreated with 5-aza-d C. After treatment with 5-aza-d C, the expression of mi R-203 a was enhanced, the methylation level of mi R-203 a in YES-2 cell line was decreased, and complete unmethylation of mi R-203 a was detected in TE1, TE13, ECa109, and T.TN cell lines. 1.2 The expression of mi R-203 a in ESCCThe expression of mi R-203 a in tumor tissues(0.253±0.099) was significantly lower than that in corresponding normal tissues(0.481±0.189)(P<0.05). The expression of mi R-203 a was correlated with status of differentiation and TNM stage of ESCC patients(t=-14.137,P=0.000),however, it was not associated with lymphatic metastasis, age, and gender(P>0.05). 1.3 The methylation status of mi R-203 a in ESCCThe promoter methylation frequency of in tumor specimens 62.7%(52/83)was significantly higher than that in corresponding normal tissues 7.2%(6/83)(χ2=3.918, P=0.048). Methylation frequencies of mi R-203 a gene in poor differentiation group 75.0%(33/44)was much higher than that in moderate and high differentiation group 48.7%(19/39)(χ2=6.103,P=0.013). Methylation frequencies of mi R-203 a in Ⅲ stage and Ⅳ stage 94.1%(16/17)was significantly higher than that in Ⅰ stage and Ⅱ stage 54.5%(36/66)(χ2=9.047,P=0.003). Methylation status of mi R-203 a gene was not associated with lymph node metastasis, age, and gender(P>0.05). 1.4 Relationship between methylation status of mi R-203 a and its expressionThe expression of mi R-203 a in tumor tissues with promoter methylation of the gene(0.232±0.818) was significantly lower than that in tumor tissues with unmethylation of the gene(0.313±0.120)(P=0.006). 2 The expression and methylation of mi R-203 b gene in ESCC 2.1The expression and methylation status of mi R-203 b in esophageal cancer cell linesRelatively low expression and hypermethylation of mi R-203 b was detected in the five esophageal cancer cell lines untreated with 5-aza-d C. After treatment with 5-aza-d C, the expression of mi R-203 b was enhanced, and the methylation status of mi R-203 b was reversed in esophageal cancer cell lines. Complete unmethylation of mi R-203 b was detected in TE1, TE13, and ECa109 cell lines. 2.2 The expression of mi R-203 b in ESCCTh expression of mi R-203 b in tumor tissues(0.026±0.021) was significantly lower than that in corresponding normal tissues(0.049±0.041)(t=7.810 P=0.000). The expression of mi R-203 b was correlated with status of differentiation of ESCC patients(P <0.05),however, it was not associated with age, gender, lymphatic metastasis and TNM stage(P>0.05). 2.3 The methylation status of mi R-203 b in ESCCThe promoter methylation frequency of in tumor specimens 57.8%(48/83)was significantly higher than that in corresponding normal tissues 12.0%(10/83)(χ2=6.673,P=0.010). Methylation frequencies of mi R-203 b gene in poor differentiation group 68.2%(30/44)was much higher than that in moderate and high differentiation group 46.2%(18/39)(χ2=4.114,P=0.043). Methylation status of mi R-203 b gene was not associated with age, gender, lymph node metastasis and TNM stage(P>0.05). 2.4 Relationship between methylation status of mi R-203 b and its expressionThe expression of mi R-203 b in tumor tissues with promoter methylation of the gene(0.017±0.013) was significantly lower than that in tumor tissues with unmethylation of the gene(0.036±0.025)(P=0.001).Conclusions:1 The relatively low expression and the hypermethylation of mi R-203 a and mi R-203 b gene were detected in the esophageal cancer cell lines. 5-aza-d C can reverse the methylation status of mi R-203 a and mi R-203 b and restore its expression in esophageal cancer cell lines, indicating that promoter exception hypermethylation of mi R-203 a and mi R-203 b gene may be an important mechanism for its inactivation in ESCC.2 The expression of mi R-203 a and mi R-203 b in ESCC tissues was significantly lower than that in the corresponding normal tissues, indicating that mi R-203 a and mi R-203 b gene may play a major tumor suppressor gene of the role in ESCC.3 The promoter methylation frequency of mi R-203 a and mi R-203 b in tumor specimens was significantly higher than that in corresponding normal tissues, indicating that promoter methylation of mi R-203 a and mi R-203 b may be associated with the occurrence of ESCC.4 The methylation status of mi R-203 a and mi R-203 b promoter was not correlated with age, gender of patients. However, it was significantly related to differentiations, indicating that promoter methylation of mi R-203 a and mi R-203 b may be associated with tumor prognosis.