Studies On Expression Of RASSF1A And Promoter Methylation Of Tumor Suppressor Genes In Laryngeal Squamous Cell Carcinoma

Activation of oncogenes and inactivation of tumor suppressor genesare required for tumorigenesis. Inactivation of tumor suppressor geneis normally due to classic mutation/deletion events and epigenticevents. Methylation is included in epigenetic events. Gene silencingby promoter hypermethylation plays an important role in thedevelopment of many cancers. In recent years, reports about epigeneticevents have been growing. RASSF1A as a new tumor suppressor gene wasidentified in chromsome 3 in 2000. RASSF1A are inactivated frequentlyby loss of heterozygosity (LOH) and promoter hypermethylation ratherthan gene mutaion in human cancers. Larynx squamous cell carcinomaaccounts for 5.7～7.6% in human cancers. In this paper we studied onpromoter hypermethylation of RASSF1A and its expression and analizedpromoter hypermethylation of FHIT and VHL.1. Promoter hypermethylation of RASSF1A gene in laryngealsquamous cell carcinoma(LSCC),corresponding noncancerous tissue andnormal blood plasma.The methylation-specific PCR was used to detect the promotermethylation of RASSF1A gene in the 48 tumor tissue, 48 correspondingnoncancerous tissue and 48 normal blood plasma and to investigate therelationship between it and LSCC. The methylation of tumor tissue(70.83%) was higher than corresponding noncancerous tissue and normalblood plasma, indicating that the promoter methylation of RASSF1A washighly associated with the carcinogenesis of LSCC. There was nosignificant correlation in the methylation of RASSF1A and tumordifferentiation, indicating that the promoter methylation of RASSF1Amight be an early event in the carcinogenesis of LSCC.2. Expression of RASSF1A in laryngeal squamous cell carcinoma(LSCC),corresponding noncancerous tissue and normal tissue.Immunohistochemistry and Western blotting were used to examine theexpression of RASSF1A in LSCC,corresponding noncancerous tissue andnormal tissue. The results of Immunohistochemistry showed that thepositive percentage of cells in LSCC was 21.49%±11.24%, 42.04%±3.34%in corresponding noncancerous tissue and 56.63%±3.86% in normaltissue. The results of Western blotting showed that the percentage ofweak positive was 56.25%(27/48) in LSCC, 16.67%(8/48) in correspondingnoncancerous tissue and 7.69%(1/13) in normal tissue, indicating theinactivation and low expression of RASSF1A may play an important rolein the carcinogenesis of LSCC. The expression of RASSF1A in stageⅢ and stage Ⅳ was significantly lower than that in stage ⅠandstageⅡ,indicating there was significant correlation between RASSF1Aexpression and tumor stage. The expression of RASSF1A in methylatedLSCC and corresponding noncancerous tissue were significantly lowerthan that in unmethylated LSCC and corresponding noncancerous tissue,so there might be highly correlation between promoter methylation ofRASSF1A gene and the expression of RASSF1A .3. Promoter hypermethylation of FHIT and VHL gene in laryngealsquamous cell carcinoma(LSCC),corresponding noncancerous tissue andnormal blood plasma.The methylation-specific PCR was used to detect the promotermethylation of FHIT and VHL gene in the 48 tumor tissue, 48corresponding noncancerous tissue and 48 normal blood plasma and toinvestigate the relationship between them and LSCC. In LSCC, the ratesof hypermethylation in FHIT and VHL were 60.42%(29/48)and 12.5%(6/48).The methylation degree of tumor tissue was higher than correspondingnoncancerous tissue and normal blood plasma, indicating that thepromoter methylation of FHIT was highly associated with thecarcinogenesis of LSCC. Concurrent methylation in RASSF1A ,FHIT andVHL were occurred in 5 samples, and among them , 4 samples in stageⅢ and stage Ⅳ;there were 19 samples occurred concurrent methylationin RASSF1A and FHIT , 12 samples in stage Ⅲ and stage Ⅳ among the19. These findings suggest that the accumulation of hypermethylationof tumor suppessor genes contributes to the tumor progression duringtumor development.More and more reports identified that tumor suppressor genes playan important role in human cancers. Promoter methylation of tumorsuppressor gene has been a focus of tumorigenesis. MSP is a fast,easyand sensitive method. As a new tumor suppressor gene, RASSF1A takespart in cell cycle suppress and regulation. As a new biological marker,promoter methylation of RASSF1A has potencial clinical benefits inearly cancer diagnosis and treatment.