| Objective Malignant tumor is a serious threat to human health.Esophageal cancer (EC) is one of common malignant tumors, and theincidence is very high in North China, especially in Cixian and Shexian ofHebei Province. There are two major histologic types of EC: esophagealsquamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC).Due to the occult symptoms, most patients are in advanced stages ofESCCwhen they have obvious symptoms, and the prognosis is poor. To reducethe morbidity and mortality of EC, it is necessary to pay great attention to thepathogenesis research. In addition to the change of the DNA nucleotidesequence may influence the occurrence and development of tumors, the role ofepigenetic mechanisms in tumor formation have received more and moreattention. DNA methylation is the most common epigenetic changes ineukaryotic cell genome. Aberrant DNA methylation can be detected both intumor tissue and blood of tumor patients, indicating that DNA methylationmay be served as a new sensitive indicator of tumor formation which may beused in the prevention and treatment of cancer.RASSF1is a tumor suppressor gene which was found in the study ofnon-small cell lung cancer by Dammann R et al in the year of2000, and thefollowing studies found that RASSF1gene may have the effect of tumorsuppressor genes by inducing apoptosis, inhibiting cell growth and migration.There are seven transcripts of RASSF1: RASSF1A-G. RASSF1D-G show thesame gene location with RASSF1A, owing the same first exon (1α) and CpGislands in promoter region. RASSF1A, RASSF1B and RASSF1C locate in thedifferent chromosome sites, and the corresponding promoter regions aredifferent. As the main transcripts of RASSF, the distribution of RASSF1A,RASSF1B and RASSF1C may be different in different tissues. Aberrant DNA hypermethylation of RASSF1A may be one of the important mechanismsleading to low expression of the gene in a variety of tumors; however, theexpression and epigenetic inactivation mechanism of the different transcriptsof RASSF1in ESCC have not been reported. In the present study, expressionof RASSF1different transcripts was detected and the main transcript wasdetermined in ESCC, and the methylation status of the main transcript wasfurther detected to provide molecular target for the prevention and treatmentof ESCC.Methods:1Reverse transcription polymerase chain reaction (RT-PCR) method wasused to detect the mRNA level of the main transcripts of RASSF1geneRASSF1A, RASSF1B and RASSF1C in48ESCC tumor tissues andcorresponding normal tissues.2Immunohistochemistry method was used to examine the proteinexpression of RASSF1A in48ESCC tumor tissues and correspondingnormal tissues.3Methylation specific PCR (MSP) method was used to examine themethylation status of RASSF1A gene in48ESCC tumor tissues andcorresponding normal tissues.4SPSS13.0was applied to analyze the results of experiments.Results:1The mRNA expression of RASSF1A, RASSF1B and RASSF1C in ESCC1.1The mRNA expression of RASSF1A in ESCCRASSF1A mRNA expression in tumor tissues (0.31±0.28) wassignificantly lower than that in corresponding normal tissues (1.1±0.65)(P=0.000). The mRNA expression of RASSF1A was not associated with age(P=0.701) and gender (P=0.730), and was associated with status of lymphnode metastasis, TNM stage and pathological differentiation: RASSF1AmRNA expression in non lymph node metastasis group (0.53±0.26) wassignificantly higher than that in lymph node metastasis group (0.08±0.05)(P=0.000), RASSF1A mRNA expression inⅠ+Ⅱ stage group (0.36±0.29) was significantly higher than that in Ⅲ+Ⅳ stage group (0.16±0.18)(P=0.034), RASSF1A mRNA expression in well and moderate differentiatedgroup (0.44±0.30) was significantly higher than that in poor differentiatedgroup (0.14±0.15)(P=0.003).1.2The mRNA expression of RASSF1B in ESCCRASSF1B mRNA expression in tumor tissues (0.60±0.28) was notsignificantly different from that in corresponding normal tissues (0.67±0.52)(P=0.533).1.3The mRNA expression of RASSF1C in ESCCRASSF1C mRNA expression in tumor tissues (0.84±0.42) was notsignificantly different from that in corresponding normal tissues (0.97±0.39)(P=0.195).2The protein expression of RASSF1A in ESCCThe protein expression of RASSF1A in48tumor specimens (22.9%,11/48) was significantly lower than that in corresponding normal tissues(83.3%,40/48)(P=0.000). The protein expression of RASSF1A wasassociated with status of lymph node metastasis, pathological differentiation,but not with age (P=0.701), gender (P=0.730), and TNM stage(P=0.552):RASSF1A protein expression in non lymph node metastasis group (36.4%,8/22) was significantly higher than that in lymph node metastasis group(11.5%,3/26)(P=0.041), RASSF1A protein expression in well and moderatedifferentiated group (40.9%,9/22) was significantly higher than that in poordifferentiated group (7.7%,2/26)(P=0.006).3The methylation status of RASSF1A in ESCCThere are two CpG islands aroud RASSF1A promoter region. Accordingto the distribution of CpG loci, we chose three different areas to study themethylation status respectively. Transcription initiation site is set as “+1”,locus1located at “-159” to “-36”, locus2located at “+103” to “+302”, locus3located at “+337” to “+494”.3.1Methylation status of locus1RASSF1A methylation frequency of this locus in tumor specimens (35.4%,17/48) was significantly higher than that in corresponding normaltissues (14.6%,7/48)(P=0.021). The methylation status of this locus wasassociated with status of lymph node metastasis, pathological differentiation,but not with age (P=0.732), gender (P=0.654) and TNM stage(P=0.117): Themethylation frequency of this locus in lymph node metastasis group (57.7%,15/26) was significantly higher than that in non lymph node metastasis group(9.1%,2/22)(P=0.000), the methylation frequency of this locus in poordifferentiated group (50%,13/26) was much higher than that in well andmoderate differentiated group (18.2%,4/22)(P=0.022).3.2Methylation status of locus2RASSF1A methylation frequency of this locus in tumor specimens(41.7%,20/48) was significantly higher than that in corresponding normaltissues (18.8%,9/48)(P=0.019). The methylation status of this locus wasassociated with status of lymph node metastasis, TNM stage and pathologicaldifferentiation, but not with age (P=0.883) and gender (P=0.875): Themethylation frequency of this locus in lymph node metastasis group (57.7%,15/26) was significantly higher than that in non lymph node metastasisgroup (22.7%,5/22)(P=0.014), the methylation frequency of this locus in Ⅲ+Ⅳ stage group (66.7%,8/12) was much higher than that in Ⅰ+Ⅱ stagegroup (33.3%,12/36)(P=0.043), the methylation frequency of this locus inpoor differentiated group (57.7%,15/26) was much higher than that in welland moderate differentiated group (22.7%,5/22)(P=0.014).3.3Methylation status of locus3RASSF1A methylation frequency of this locus in tumor specimens (50%,24/48) was significantly higher than that in corresponding normal tissues(18.8%,9/48)(P=0.003). The methylation status of this locus was associatedwith status of lymph node metastasis, TNM stage and pathologicaldifferentiation, but not with age (P=0.771) and gender (P=0.755): Themethylation frequency of this locus in lymph node metastasis group (69.2%,18/26) was significantly higher than that in non lymph node metastasis group(27.3%,6/22)(P=0.004), the methylation frequency of this locus in Ⅲ+Ⅳ stage group (83.3%,10/12) was much higher than that in Ⅰ+Ⅱ stage group(38.9%,14/36)(P=0.008), the methylation frequency of this locus in poordifferentiated group (73.1%,19/26) was much higher than that in well andmoderate differentiated group (22.7%,5/22)(P=0.001).4Relationship between methylation status of RASSF1A and its expression4.1Relationship between mRNA and protein expression of RASSF1AThe mRNA expression of RASSF1A in tumor tissues with positiveprotein expression of the gene (0.49±0.29) was significantly higher than thatin tumor tissues with negative protein expression of the gene (0.04±0.03)(P=0.010).4.2Relationship between methylation and expression of RASSF1A4.2.1Locus1: The mRNA expression of RASSF1A in tumor tissues withhypermethylation of the gene (0.16±0.16) was significantly lower than that intumor tissues with unmethylation of the gene (0.47±0.32)(P=0.019). Thepositive protein expression of RASSF1A in tumor tissues withhypermethylation of the gene (23.5%,4/17) was not significantly differentfrom that in tumor tissues with unmethylation of the gene (22.6%,7/31)(P=1.000).4.2.2Locus2: The mRNA expression of RASSF1A in tumor tissues withhypermethylation of the gene (0.18±0.18) was significantly lower than that intumor tissues with unmethylation of the gene (0.44±0.33)(P=0.007). Thepositive protein expression of RASSF1A in tumor tissues withhypermethylation of the gene (10.0%,2/20) was not significantly differentfrom that in tumor tissues with unmethylation of the gene (32.1%,9/28)(P=0.147).4.2.3Locus3: The mRNA expression of RASSF1A in tumor tissues withhypermethylation of the gene (0.18±0.17) was significantly lower than that intumor tissues with unmethylation of the gene (0.44±0.32)(P=0.006). Thepositive protein expression of RASSF1A in tumor tissues withhypermethylation of the gene (8.3%,2/24) was significantly lower than that intumor tissues with unmethylation of the gene (37.5%,9/24)(P=0.016). Conclusions:1The expression of RASSF1A in ESCC tumor tissues was significantlylower than that in the corresponding normal tissues, but the expression ofRASSF1B and RASSF1C in ESCC tumor tissues was not significantlydifferent from that in corresponding normal tissues.2The decreased expression of RASSF1A may be associated with theoccurrence of ESCC, and DNA hypermethylation of RASSF1A promoter andthe first exon may be one of the mechanisms leading to inactivation of thegene in ESCC.3The methylation frequencies of different CpG loci in RASSF1A genepromoter region and the first exon region were distinct, indicating themethylation of critical CpG loci may be one of the main mechanisms leadingto the decreased expression of the gene in ESCC.4The methylation status of RASSF1A had no association with age andgender of ESCC patients. However, it was significantly related to status oflymph node metastasis, TNM stage and pathological differentiation, indicatingthat methylation of RASSF1A may be associated with the occurrence,development and prognosis of ESCC. |
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