Transcriptional Expression And Promoter Hypermethylation Of RASSF1A In Oral Premalignant Lesions And Squamous Cell Carcinomas

Oral squamous cell carcinoma (OSCC) is one of the most common oral cancers, which is a serious of threat to human health with a very high morbidity and a low viability. Carcinogenesis must be understood in terms of accumulation of mutation in regulatory genes, including activation of oncogenes and inactivation or loss of tumor suppressor genes. As a result, gene diagnosis and gene therapy have been paid more attention day by day. Great deal of findings suggested that inactivation of the tumor suppressor genes at 3p should be an early and critical event in the development of many cancers. Previous research has noticed that Heterozygosity (LOH) and deletion of gene fragment in 3p is a common Phenomenon in mang types of malignant tumors.Those predict that this region has tumor suppressor gene. In 2000, Dammann et al , isolated a RAS association domain family l, RASSF1, which was located in the 120-kb region of minimal homozygous deletion at 3p21.3 in lung cancer. The RASSFI gene encoded three major transcripts, RASSFIA, RASSF1B and RASSFIC, which was produced by alternative promoter selection and different message RNA(mRNA) splicing. RASSFIA was frequently inactivated in lung cancer cell lines by promoter hypermethylation. However mutation was rarely observed. Reexpression of this gene in lung cancer cell lines reduced colony formation, suppressed anchorage-independent growth, and inhibited tumor formation in nude mice. Therefore, RASSFIA was supposed to be one of new candidate tumor suppressor genes in lung cancer. There are only a few researches of RASSF1A in OSCC. In the present study,we used RT-PCR to evaluate the transcriptional expression of RASSF1A and used MSP (Methylation-specific PCR) method to detect the methylation status of RASSF1A promotor CpG island in the same specients from DNA level in oral premalignant lesions and SCC to clarify the possible role of RASSF1A in oral stratifid squamous epithelial tumors. Results: In this study, firstly we used RT-PCR to evaluate the transcriptional expression of RASSFIA. The results showed that RASSF1A transcripts were expressed in all normal oral mucosa (NOM) tissues. The loss transcriptional expression of RASSFIA mRNA 25%(5/20) of oral premalignant lesions and in 46.88% (15 of 32) SCC. The loss transcriptional expression of RASSFIA was up-regulated in SCC comparing with the NOM and prmaligantlesions(p<0.05). The loss transcriptional expression of RASSFIA of SCC was higher in poor diffrentiated SCC than in well-differentiated SCC(p<0.05). The expression of RASSF1 mRNA was not associated with T, N, , TNM stage,sex or smoking. Meanwhile, we used MSP (Methylation-specific PCR) method to detect the methylation status of RASSFIA promotor CpG island in the same specimens from DNA level. MSP analysis demonstrated that 15% (3/20) of oral premalignant lesions was hypermethylated at the CpG island in the promoter of RASSFIA. And so 40.63% of SCC is. Further, non-expressing SCC usually methylated at high level. But none of NOM tissues was hypermethylated at the CpG island in the promoter of RASSFIA. Significant diference of RASSFIA hypermethylation was observed between SCC and NOM (p<0.05). The promoter hypermethylation of RASSFIA of poor differentiated SCC was higher in SCC (p<0.05). The promoter hypermethylation of RASSF1A was not associated with T, N, TNM stage,sex or smoking. DNA methylation is an important regulator of gene transcription, and its role in carcinogenesis has been a topic of considerable interest in the last few years. Alterations in DNA methylation are common in a variety of tumors as well as indevelopment. Of all epigenetic modifications, hypermethylation, which represses transcription of the promoter regions of tumor suppressor genes leading to gene silencing, has been most extensively studied. However, global hypomethylation has also been recognized as a cause of oncogenesis. New information oncerning the mechanism of methylation and its control has led to the discovery of many regulatory proteins and enzymes. The contribution of dietary folate and methylene terahydrofolate reductase polymorphisms to methylation patterns in normal and cancer tissues is under intense investigation. As methylation occurs early and can be detected in body fluids, it may be of potential use in early detection of tumors and for determining the prognosis. Because DNA methylation is reversible, drugs like 5'-azacytidine, decitabine, and histone deacetylase inhibitors are being used to treat a variety of tumors. Novel demethylating agents such as antisense DNA methyl transferase and small interference RNA are being developed, making the field of DNA methylation wider and more exciting RASSF1A gene has 1873 bp, and encodes protein with 340 amino acids. According to its protein sequence,their function could...