Expression And Epigenetic Inactivation Mechanism Of RASSF5Different Transcripts In Esophageal Squamous Cell Carcinoma

Objective: Esophageal cancer is one of the common malignant tumors ofhuman, and China is a country with high incidence regions of esophagealcancer. The north districts of China especially along the Taihang Mountains ofHebei, Henan Province are high incidence regions of esophageal cancer, andesophageal squamous cell carcinoma (ESCC) is the most common type inthese districts. Due to the occult symptoms, most patients are in advancedstages of ESCC when first visit their physicians, and quite a few patients cannot take surgical treatment and the prognosis is poor. It is necessary toimprove the early diagnosis of esophageal cancer and to strengthen the studyon etiology for primary prevention. It is generally considered that esophagealcancer is a multi-factorial, polygenic, and several stages of developmentprocess, its etiology and pathogenesis has not been fully clarified yet.In addition to the change of the DNA nucleotide sequence may influencethe occurrence and development of tumors, the role of epigenetic mechanismsin tumor formation have received more and more attention. As an importantpart of epigenetics, DNA methylation is one of the first discovered epigeneticmodifications and may play significant roles in maintaining normal cellularfunction, embryonic development and tumorigenesis. DNA methylationalterations of tumor-related genes ramain widespread in tumorigenesis, and therole of DNA methylation in the development and progression of tumors is thehot spot of current research. Investigations have found that the development oftumors might be associated with the inactivation of tumor suppressor geneswhich caused by the aberrant methylation of CpG island in the promoter. As amember of the Ras-Association Domain Family, RASSF5(often calledNORE1, Novel Ras Effector1) is the human homologue of the mouse Raseffector Nore1. RASSF5gene is located at1q32.1. As a result of alternative splicing and differential promoter usage, RASSF5contains threedifferent transcripts, termed RASSF5A, RASSF5B and RASSF5C. The threetranscripts contains different exons, RASSF5A comprises exons1,2,4,5,6,7,RASSF5B comprises exons1,2,4,5,7, and RASSF5C comprises exons3,4,5,6,7. The role of different transcripts of RASSF5may be different in ESCC. Inthe present study, expression of RASSF5different transcripts were detectedand the main transcript was determined in ESCC, and the methylation status ofthe main transcript was further detected in esophageal cancer cell lines andtissues of ESCC to explore the role of different transcripts of RASSF5inESCC.Method:1Reverse transcription polymerase chain reaction (RT-PCR) method wasused to detect the mRNA level of RASSF5different transcripts in ESCCtumor tissues and corresponding normal tissues, and in esophageal cancercell lines (TE1, TE13, T.TN, Ec109) treated or untreated with5-aza-dC.2Methylation specific PCR (MSP) method was used to examine themethylation status of RASSF5A gene in esophageal cancer cell linestreated or untreated with5-aza-dC,49ESCC tumor tissues andcorresponding normal tissues.3Immunohistochemistry method was used to examine the proteinexpression of RASSF5A in ESCC tumor tissues and corresponding normaltissues.4SPSS13.0was applied to analyze the results of experiments.Results:1The mRNA expression of RASSF5different transcripts in esophagealsquamous epitheliumRASSF5A was the main transcript that expressed in esophagealsquamous epithelium, however, RASSF5B and RASSF5C were expressedvery low.2The mRNA expression and methylation status of RASSF5A in esophagealcancer cell lines 2.1The mRNA expression of RASSF5A in esophageal cancer cell lines treatedor untreated with5-aza-dCTE1, Ec109cell lines showed weak positive staining, and TE13, T.T Ncell lines showed no expression of RASSF5A. When the cell lines weretreated with5-aza-dC, mRNA expression level of RASSF5A in TE1, Ec109cell lines was higher, and mRNA expression of RASSF5A can be detected inTE13, T.T N cell lines.2.2The methylation status of RASSF5A in esophageal cancer cell linestreated or untreated with5-aza-dCRASSF5A gene was fully methylated in TE13, T.TN, Ec109cell lines,while semi-methylation of RASSF5A was detected in TE1. After treatmentwith5-aza-dC, RASSF5A gene was detected unmethylation status in TE13,T.TN, and Ec109cell lines, and the methylation level of RASSF5A in TE1cellline was decreased, the unmethylation level of RASSF5A in TE1cell line wasincreased.3The relationship between mRNA and protein expression, and methylationstatus of RASSF5A and ESCC clinical pathological data3.1The mRNA expression of RASSF5A in ESCCRASSF5A mRNA expression in tumor tissues (0.57±0.18) wassignificantly lower than that in corresponding normal tissues (0.87±0.10)(t=-10.201, P=0.000). The mRNA expression of RASSF5A was correlatedwith status of lymph node metastasis, pathological differentiation and TNMstage of ESCC patients (P <0.05), however, it was not associated with age andgender (P>0.05).3.2The protein expression of RASSF5A in ESCCThe protein expression of RASSF5A in tumor specimens (26.5%,13/49)was significantly lower than that in corresponding normal tissues (75.5%,37/49)(P=0.000). The protein expression of RASSF5A was significantlyassociated with status of lymph node metastasis and pathologicaldifferentiation of ESCC (P<0.05), however, it was not associated with age,gender and TNM stage (P>0.05). 3.3The methylation status of RASSF5A in ESCCThe promoter methylation frequency of RASSF5A in tumor specimens(55.1%,27/49) was significantly higher than that in corresponding nomaltissues (28.6%,14/49)(P=0.011). Methylation status of RASSF5A gene wasnot associated with age, gender and TNM stage (P>0.05). Methylationfrequencies of RASSF5A gene in poor differentiation group (75.9%,22/29)was much higher than that in moderate and high differentiation group (25.0%,5/20)(χ2=12.377, P=0.000). Methylation frequencies of RASSF5A in lymphnode metastasis group (80.8%,21/26) was significantly higher than that in nonlymph node metastasis group (26.1%,6/23)(χ2=14.750, P=0.000).3.4Relationship between methylation status of RASSF5A and its expressionThe mRNA expression of RASSF5A in tumor tissues with positiveprotein expression of the gene (0.68±0.17) was significantly higher than thatin tumor tissues with negative protein expression of the gene (0.52±0.16)(t=-3.036, P=0.007). The mRNA expression of RASSF5A in tumor tissueswith promoter hymethylation of the gene (0.51±0.16) was significantly lowerthan that in tumor tissues with unmethylation of the gene (0.64±0.17)(t=-2.737, P=0.009). The positive protein expression of RASSF5A in tumortissues with promoter hymethylation of the gene (11.1%,3/27) wassignificantly lower than that in tumor tissues with unmethylation of the gene(45.5%,10/22)(χ2=7.335, P=0.007). RASSF5A protein expression wasinversely correlated with its promoter methylation status (R=-0.387, P=0.006).Conclusions:1The main transcript of RASSF5which was expressed in esophagealsquamous epithelium maybe RASSF5A.2Methyltransferase inhibitor5-aza-dC can reverse the methylation status ofRASSF5A and restore its expression in esophageal cancer cell lines,indicating that promoter exception hypermethylation of RASSF5A genemay be an important mechanism for its inactivation in ESCC.3The promoter methylation frequency of RASSF5A in tumor specimenswas significantly higher than that in corresponding nomal tissues, indicating that promoter methylation of RASSF5A may be associated withthe occurrence of ESCC.4The methylation status of RASSF5A promoter was related to pathologicaldifferentiation and lymph node metastasis, indicating that promotermethylation of RASSF5A may be closely related to metastasis of ESCC.