The Experiment Study Of Fragile Histidine Triad In Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma(OSCC)is one of the most common malignancies in our country,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. But now its mechanism of the development and progression remains unclear so far.It is helpful for us to find and identify new genes responding for OSCC so as to reveal the molecular mechanism and genetic susceptibility of OSCC.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 many types of malignant tumors.Those predict that this region has tumor suppressor gene.Fragile histidine triad(FHIT)gene lies in the chromosome 3p14.2 and belongs to HIT gene family.It is the first antioncogene which associates the fragility site with tumor.It can suppress tumor probably by inducing cell apoptosis.There exist hypermethylation, abnormal transcription of FHIT gene and down regulation expression of FHIT protein in liver OSCC.The alternations of FHIT gene correlate with several clinic pathological indicators such as progression,invasion,relapse rate,and survival rate of OSCC.The alternation of FHIT gene is an early and frequent event of OSCC,and FHIT gene as a new molecular indicator of oral cancer canmonitor.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 in development.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.Objective To study the expression and significance of fragile histidine triad(FHIT)in oral squamous cell carcinoma(OSCC)But the reports of the deletion and mutation of FHIT gene in OSCC,pleomorphic adenoma and mucoepidermoid carcinoma of parotid gland are few.To detect the mRNA expression of FHIT in OSCC and to investigate the relationship between FHIT gene and the occurrence and development of OSCC,we detected the mutation and deletion of exon5,and exon8 of FHIT gene by PCR,and discuss if there are homozygous in exon5 and exon8 of FHIT gene in OSCC.To detect FHIT gene aberrant methylation and its expression in OSCC so as to investigate its role in tumor genesis and progression of OSCC.Our study will lay the foundation for further investigation of the relationship between FHIT gene and oral squamous cell cacinomas.Methods We adopted immunohistochemicalmethed in the study to investigate the expression of FHIT in 48 cases of OSCC and 26 cases of normal oral mucosa.The data was analyzed by Chi-square test.With reference to the expression ofβ-actin,the expression of FHIT mRNA was determined by reverse transcription polymerase chain reaction(RT-PCR)in 48 OSCC cases and 26 normal tissue,and Semi-quantitative analysis of band densities was performed..We collected 48 OSCC cases and 26 normal tissue DNA was extracted from these tissues.The method of polymerase chain reaction(PCR) was set up to analyze the homozygous deletion of exon5 and exon8 of FHIT. We used RT-PCR to evaluate the transcriptional expression of FHIT and used MSP(Methylation-specific PCR)method to detect the methylation status of FHIT promotor CpG island in the same specients from DNA level in oral premalignant lesions and SCC to clarify the possible role of FHIT in oral stratifid squamous epithelial tumors.Results Positive rate of FHIT in OSCC 43.75%(21/48)was lower than that in normal oralmucosa)76.92%(20/26)The difference was statistically significant(P＜0.05)The expression of FHIT was not significantly correlated with age and sex of patients(P＞0.05),but FHIT protein content was significantly associated with differentiation(P＜0.05),the poorer the differentiation,the less the expression.The mRNA expression of FHIT gene was significantly decreased in OSCC tissues compared with in normal oral mucous membrane.There was homozygous deletion in exon5 and exon8 of FHIT gene in parts of OSCC,after PCR amplification.There was no mutation in exon5 and exon8 of FHIT gene in OSCC,We analysis the cDNA transcrips, In addition,we simplified the sequencing method in our experiment.We are sequencing the purified production of PCR directly in this study.To discover in the abnormal transcrisp 350bp or so,there absence exon5-exon8.it only connected exon4 with exon8.We haven't detected the methylation status of FHIT promotor CpG island in the normal specimens from DNA level.The results showed that significant diference of FHIT hypermethylation was observed between SCC and NOM(p＜0.05).The promoter hypermethylation of FHIT of OSCC was higher in NOM(p＜0.05). Conclusions Loss of FHIT mRNA expression occurs increasingly commonly during the genesis and development of OSCC.The incidence of negative expression of FHIT mRNA is higher in poorly differentiated OSCC. FHIT may help the identification of OSCC's pathologic grade.Our study suggests that detection of FHIT hypermethylation by MSP method can provide important evidence for molecular staging and molecular pathology. FHIT could be a new useful molecular marker of OSCC gene diagnosis and gene therapy of oral carcinoma.So FHIT may play an important role in gene diagnosis and gene therapy of oral carcinoma.