The Research Of CDH1 Methylation And Mutations In The Process Of SD Rats Tongue Carcinogenesis

Background:Oral squamous cell carcinoma(OSCC)is the most frequently oral malignancy,accounting for 90 percent of oral cancer.OSCC has a higher degree of malignancy,its 5-year survival rate is only about 50%,and it is a serious threat to human health.It occurs in the tongue mucosa that are known as tongue squamous cell carcinoma(TSCC).E-cadherin(epithelial cadherin,E-cad)is an adhesion molecule,and is closely related with tumor.It is a kind of calcium-dependent transmembrane glycoprotein.Under the effect of different connexins,which can be connected to different cytoskeletal components.Intracellular domain of E-cadherin constitutes the core functional unit by a,β-catenin and p120-catenin.After binding with a number of related proteins,which is connected to the actin cytoskeleton.Or α-catenin directly is connected with the fibrous actin.E-cadherin gene CDH1 locates in the long arm of chromosome 16(16q22.1).Its DNA is about 100Kb,and consists of 16 exons and 15 introns,which contains 2.6kb coding sequence.Intercellular adhesion molecules in ontogeny,signal transduction,gene regulation,cell proliferation and development plays an important role,and associated with many cancers.Currently,many scholars are conducting research in this area,In particular,the studies of ovarian cancer,breast cancer,stomach cancer and other are more in-depth,The study found that E-cadherin adhesion molecule expression is reduced in human lung cancer,gastric cancer,breast cancer,ovarian cancer,prostate cancer and other tumors,and this phenomenon is universal.E-cadherin is the main elements of keeping the epithelial cells adhere to each other in adult vertebrates.In a variety of tumor tissues,E-cadherin decreases or disappeares in the cell surface,cause cancer cells easily detach from the tumor mass,and this become one of the possible reasons for the invasion and metastasis.This study focused on the CDH1 promoter methylation and mutations in exon at various pathological stages of tongue mucosa carcinogenesis of SD rat.And explore the molecular mechanisms of tongue mucosa carcinogenesis by 4NQO drinking induced in animal models.Objective:Research CDH1 gene methylation and mutation in exon at various pathological stages of tongue mucosa carcinogenesis.Provide a theoretical basis for exploring regulatory role of CDH1 gene in the process of formation of tongue squamous cell carcinoma.Materials and Methods:1.75 SD rats were administered 0.004%4-nitroquinoline-l-oxide(4NQO)in drinking water from the age of 6 weeks to the age of 30 weeks,15 SD rats were kept in the same conditions without 4NQO,to act as negative controls.Killed the rats at different stages.Tumors were removed from the surrounding mucosa and cut into four pieces,one of which underwent histological examination,and the others were kept frozen.2.Using DNA extraction kit to extract DNA from frozen tongue tissue of control group SD rats and experimental group SD rats.These tissues include normal tongue mucosa,epithelial hyperplasia tongue mucosa,mild epithelial dysplasia tongue mucosa,moderate epithelial dysplasia tongue mucosa,severe epithelial dysplasia tongue mucosa and tongue squamous cell carcinoma.3.The genomic DNA of normal SD rats tongue mucosa and tongue mucosa in various pathological stages of tongue carcinogenesis act as research subjects,SD rat methylated genomic DNA acts as positive control,deionized water acts as the blank control,designed specific methylated and unmethylated primers of CDH1 promoter CpG island,applied methylation specific PCR(Methylation-Specific PCR,MS-PCR)to detect the different levels of CDH1 promoter CpG island methylation in the various stages of tongue squamous cell carcinoma.4.The genomic DNA of SD rats tongue mucosa in various pathological stages of tongue carcinogenesis act as research subjects,the normal SD rats tongue mucosa genomic DNA act as negative control,designed the forward and reverse primers of CDH1 exons 1-16,and used polymerase chain reaction(PCR)amplification of these exons,then sequenced them after purification to.detect CDH1 exons mutations in the various stages of tongue mucosa carcinogenesis.Results:1.Eliminate died while to establish animal model of rats,pathological changes of experimental rats and losses in the process of experiment,end up with 62 cases of SD rat tongue mucosa tissue genomic DNA,including 15 cases in the control group,15 cases of epithelial hyperplasia,10 cases of mild dysplasia,8 cases of between moderate and severe dysplasia,14 cases of squamous cell carcinoma.2.MS-PCR results showed that in different stages of the SD rat tongue mucosa cancer has been CDH1 promoter CpG island methylation specific product,but can not detect the CDH1 promoter methylation.3.All samples sequenced exons 1-16 showed that only exon 13 had mutation,it occurred in the site of mRNA 2106(G→T).It occurred 1 case in moderate and severe dysplasia group(12.5%)and 5 cases in cancer group(35.71%),cancer group and control groups,simple hyperplasia group,mild dysplasia group 3 groups compared with the difference was statistically significant(P<0.05),the relation between abnormal epithelial hyperplasia group,normal group and tongue squamous carcinoma group,comparing the two groups have no statistical difference(P>0.05).At the same time with the increasing of degree of pathological grading,CDH1 exons mutation rate showed a trend of increase(P<0.01).Conclusions:In the model of SD rats tongue squamous cell carcinoma induced by 4NQO,CDH1 promoter CpG island did not occur methylation,however,there had detected a suspected missense mutation in CDH1 exon 13 in experimental groups.Occurrence and development of tongue squamous cell carcinoma of SD rats which induced by 4NQO drinking act may be associated with the CDH1 mutations in exons,but with the CDH1 promoter methylation is independent.