The Investigation Of Microsatellite Instability And Loss Of Heterozygosity In Laryngeal Squamous Cell Carcinomas

Laryngeal carcinoma is frequently seen in head and neck malignant neoplasms, accounting for 1%-5%of human cancers. In recent years, with the change of the environmental factors, such as smoking and drinking, the tendency of its morbidity and mortality is increasing, and seriously hampers the health and life of patients. So it is important to study carcinogenesis and reveal etiopathogenesis of laryngeal carcinoma. Like other malignant tumours, laryngeal carcinoma is due to not only environment but also heredity factors. Detection of microsatellite alterations is a hot point of genetic research.The aim of our investigation is to study the incidence of microsatellite instability (MSI) and loss of heterozygosity (LOH) in the laryngeal squamous cell carcinomas at the regions of 3p14.1﹑5q23.1﹑9p22.1 and 11p15.4, Then disscuss the relationship between the microsatellite alternations and the clinical informations of the patient with tumours. After that we can make an estimate that if the microsatellite alternation take part in the pathogenic mechanism of in laryngeal squamous cell carcinomas. Microsatellite which is noncoding and generally reside in genome of human being is a kind of simple and short sequence repeats with high polymorphism. It is excellent in the aspects of numbers,stability and distribution and controls the expression of gene by combineing with some specific proteins or change the conformation of genes. MSI and LOH are the commom phenomenons in malignant tumors. MSI makes us know the anomaly of mismatch repair gene and destabilizes the genome, then the expression and regulation of genome are inordinate, cellular proliferation precipitates the formation of tumors. LOH is often seen in the place nearby anti-oncogene. The loss of genome can lead to bad expression of gene or disfunction of protein. The functions of suppressing cellular proliferation and induceing cellular apoptosis are lost. The effectiveness of oncogenes enhance and makes cells generate and differentiate. Even vascularization induces the infiltration and metastasis of cancer. So it can be use to decide whether the genome is steady or the tumor-suppressing gene participate in carcinogenesis to compare the carcinomas with tissues around by the microsatellite alternation. Microsatellite alternation has already be used to investigate many malignant tumours. MSI was firstly discovered in the research of colon carcinomas. After that LOH and MSI was also detected in carcinomas of stomach,lung,liver,esophagus,breast and leucocythemia. The studies of cancer related gene are also can be seen in the laryngeal squamous cell carcinomas. The genes which often have effectives in head and neck carcinomas contain FHIT and hMLH1 on the chromatosome of 3p,APC on the chromatosome of 5q,p16 on the chromatosome of 9p,p53 at the domain of 17p,DDC at the domain of 18q,ING1 at the domain of 13q,IGF2 and H19 at the domain of 11p and so on. Most of them are anti-oncogenes, and most of microsatellite alternations around these genetic locus are LOH.We select four microsatellite markers :they are D5s592 (around APC)﹑D3s1228 (close to FHIT)﹑D9s162 (nearby p16)﹑D11s1758 (surrounding IGF2 and H19).They are located on the chromatosomes which are easily affecte in laryngeal squamous cell carcinomas, and most of them verge on anti-oncogene known or candidate. The cases which were proved to be the squamous cell carcinomas of larynx are analyzed by comparing tumorous tissues and tissues around with the four microsatellite markers,useing PCR and PGE–AgNO3 staining. Compared with tissues beside of carcinomas, if the electrophoresis strips of tumor tissue are lost or relative density is reduced by more than 50%, it is defined as LOH; if the electrophoresis strips of cancer tissue are increaced or displaced, it is called MSI.As far as our experimental result goes, among the 40 cases of laryngeal squamous cell carcinomas,high frequency of microsatellite abnormal occurred at D5s592. The incidence rate of LOH is 40%, while MSI is 30%, the total mutation rate of this site is 70%. Then the mutation rate of D3s1228 is 52.5%, the incidence rates of MSI and LOH are 40%and 12.5% respectively. The positive rate of D9s162 is lower, the incidence rates of LOH is 42.5%, and MSI is 5%, overview the mutation rate of D9s162 is 47.5%. The microsatellite alternation of D11s1758 is only 20%, 5%and 15% incidences of MSI and LOH are discoved. LOH of D5s592 relates to the clinical stage and patho-grade(p<0.05), It has a remarkable difference between T₁ and T₃,T₄stages, T₁ stage has a higher incidence of LOH; it also has a distinctness betweenâ…¡andâ…¢grade( p<0.05),â…¡grade has a higher mutation rate of LOH. So LOH of D5s592 is considered to be the early event of the development in laryngeal squamous cell carcinomas,and let us know that loss of APC participate in carcinogenesis, maybe through the change of aignal transduction,regulation of cell taction and cell sequencing death. Loss of APC result in the dysfunction of inhibiting cellular proliferation, and then lead to the malignant transformation of cells. MSI of D3s1228 relates to the clinical stage and patho-grade(p<0.05), it has a dramatical difference between T₁ and T₃,T₄stages, T₃,T₄ stage has a higher incidence of MSI; it also has distinctness betweenâ… andâ…¢grade( p<0.05),â… grade has a higher mutation rate of MSI . MSI of D3s1228 is think to be the terminal event in carcinomas of larynx,but the cases which are positive of MSI have low malignancy. MSI of D3s1228 remind us that loss of FHIT take part in carcinogenesis. Loss of FHIT lead to abnormal expression of gene products and reduction of gene function, inhibit the activity of hydrolytic enzyme. Through this way, the transmission of growth signals is increase, the pathway of restraint and apoptosis is cut down, then bring on carcinomas. LOH of D9s162 only relates to the patho-grade of tumors(p<0.05), it has a distinctness betweenâ…¡andâ…¢grade(p<0.05). The cases which are gradeâ…¡have low incidence of LOH. From the research, we can suppose that loss of p16 participate in carcinogenesis. p16 regulates the cycle of cell division. The loss of p16 protein can lead to disorders of generation cycle G1, Cell proliferates abnormally and tumours occurrent. Microsatellite alternation of D11s1758 dosen't relate to the clinical stage and patho-grade, but we could not conclude that no potential tumor suppressor genes present.Our studies indicate that microsatellite alternation (include MSI and LOH)is a common genetic change in laryngeal squamous cell carcinomas, and revealed that tumor related gene nearby take part in the occurrence,development,even infiltration and metastasize of tumors. Changes of microsatellite can help to direct clinical diagnosis and therapy. Details of the carcinogenesis are to be proven by further discussion. The relationship between microsatellite alternation and linked gene expression,the function of protein and its mechanisms in tumours will be the hot point of the new aspects in the future.