Study On The Expression Of Ep-CAM During The Rat Tongue Carcinogenisis Induced By 4NQO

Objective:This paper aims to obtain the lesions at different stages in the process of tongue mucosal carcinogenesis by establishing an animal model of the tongue squamous cell carcinoma(TSCC)from rats.Development and significance in the relationship between Ep-CAM and TSCC have been explored after the test for the expression on the protein and mRNA of epithelial cell adhesion molecule(Ep-CAM).This therefore provides the basis for the early diagnosis and treatment of TSCC.Methods :(1)A rat TSCC model was induced by oral administration of 4-nitroquinoline 1-oxide(4NQO)in drinking water: 84 6-week-old male Sprague-Dawley(SD)rats were randomly divided into 7 groups,including experimental groups A,B,C,D,E and F as well as control group G(each group 12 rats).The experimental group was given the 4NQO solution of 0.004%(mixed with pure sterile water,lucifugal),and the control group was given pure sterile water for drinking.These SD rats were sacrificed at 12 weeks(A),14 weeks(B),16 weeks(C),18 weeks(D),20 weeks(E)and 22 weeks(F,G).The lesions of the tongue mucosa were divided into two parts.One was fixed in 4% paraformaldehyde solution for HE staining and immunohistochemistry,another was placed in the enzyme-free cryopreservation tube containing RNAstore preservation solution keeping in-20? for the detection of quantitative PCR.Meanwhile,take lymph nodes of the neck,esophagus,lung and liver tissues for HE staining and pathological examination.(2)The expression level of Ep-CAM on protein in different pathological grading lesions of tongue mucosa from these rats was detected by immunohistochemistry.(3)The expression level of Ep-CAM on mRNA in different pathological grading lesions of tongue mucosa from these rats was detected by fluorescence quantitative real-time PCR.Results:(1)TSCC was induced in rats success and total of 80 effective samples were obtained.These specimens of tongue mucosa were divided into 6 grade according to the histology appearance about epithelial: 18 samples of normal,9 samples of simple hyperplasia,11 samples of mild dysplasia,12 samples of moderate dysplasia,12 samples of severe dysplasia,18 samples of TSCC.(2)Detecting the expression of Ep-CAM in these tissues of tongue mucosa by immunohistochemistry.The positive rate of Ep-CAM for each grade,11.11%(2/18)in normal mucosa,22.22%(2/9)in simple epithelial hyperplasia,36.37%(4/11)in mild epithelial dysplasia,66.67%(8/12)in moderate epithelial dysplasia,91.67%(11/12)in severe epithelial dysplasia,88.89%(16/18)in TSCC.The expression level of Ep-CAM was up-regulated in the progression of TSCC in rats,and the difference was statistically significant(P <0.05).Positive sites are mainly located in the membrane and cytoplasm of the epithelial cell.(3)Result from these 80 samples detected by RT-qPCR,we found that the expression level of Ep-CAM on mRNA was up-regulated in the progression of TSCC in rats.Compared with the normal group,there was no difference about the expression of Ep-CAM on mRNA in simple epithelial hyperplasia and mild epithelial dysplasia group.But the moderate,severe epithelial dysplasia and TSCC group were significantly higher than that in normal mucosa group(P <0.05).The expression of Ep-CAM mRNA in the mild,moderate,severe epithelial dysplasia and TSCC group were significantly higher than that in simple epithelial hyperplasia group(P <0.05).Compared with mild epithelial dysplasia group,the expression of Ep-CAM on mRNA in moderate,severe epithelial dysplasia and TSCC group was significantly higher(P <0.05).The expression level of Ep-CAM on mRNA in epithelial hyperplasia,mild epithelial dysplasia,moderate epithelial dysplasia,severe epithelial dysplasia,TSCC tissue were 0.79 times,1.12 times,1.82 times,2.51 times,4.45 times of that in normal tongue mucosa tissues.Conclusion:(1)By drinking water with 4-nitroquinoline-1-oxide(4NQO),TSCC could induced targeting in SD rats successfully.(2)Expression levels of Ep-CAM on protein and mRNA are significantly up-regulated in the progression of tongue mucosal carcinogenesis in rats,and they are closely related to the pathological grading of these lesions.The up-regulated level of Ep-CAM on mRNA maybe is the early event in the genesis and progression of TSCC.(3)Ep-CAM involved in the in the genesis and progression of TSCC,and it can be used as an early indicator of effective diagnosis of TSCC.Ep-CAM is expected to become an effective target for the treatment of TSCC.