| Objective:Oral squamous cell carcinoma accounts for the sixth most common mortality-related malignant cancer which causes approximately 130000 patients death worldwide.The reason is that the clinical diagnosis of oral squamous cell carcinoma is difficult,especially for early stages diagnosis of oral squamous cell carcinoma.Research shows that early oral squamous cell carcinoma patients 5 year survival rate in 80%of the cases,in patients with advanced oral squamous cell carcinoma 5 year survival rate dropped to 30%,unfortunately,there are still 2/3 patients were diagnosed as oral squamous cell carcinoma is already late,resulting in 5 year survival rate of less than 50%,Therefore,we are urgently need to explore the methods for early diagnosis of oral squamous cell carcinoma.In recent years,researchers have been detailed understanding and researching of the molecular mechanisms underlying oral squamous cell carcinoma tumourigenesis,which hope to improve the early stages diagnosis of oral squamous cell carcinoma.Tongue squamous cell carcinoma is one of the major type oral cancers,and study of the related factors in the pathogenesis is more and more in-depth.Voltage-gated potassium channels are the most common type of potassium channels on the cell membrane.The study found that Voltage-gated potassium channels are abnormal expression in many tumor cell lines.Kv3.4 is widely distributed on the cell membrane and cytoplasm of the human body,and plays an important role in regulation of cellular microenvironment.The positive expression of Kv3.4 was closely related to the occurrence of oral squamous cell carcinoma Epidermal growth factor receptor which is a receptor protein tyrosine kinase and could regulate many cellular functions,Including cell proliferation and survival.EGFR targeted therapy has been widely used in the treatment of human cancer.It has been found in study that abnormal expression of EGFR is closely related to the occurrence and development of oral squamous cell carcinoma,and it can be used as an important index for the judgment of prognosis of cancer patients.Caveolae is a small structure existing in a variety of cell surface.Caveolin-1 is the major structural protein of caveolae.The study found that it plays an important role in cell processes,including cell adhesion,molecular transport,and signal transduction.Thus,Caveolin-1 could be a key molecule for growth-related signaling and in the development of cancerMethods:1.In standard barrier environment,4NQO water-drinking method was used to induce squamous cell carcinoma of 84 SD rats tongue,and the tongue mucosal tissue samples are collected.The collected tissues are divided into two parts.One part of samples is with HE staining and immunohistochemical staining,and pathological grade was determined according to the results of HE staining(blinded by two experienced pathologists);another part of the tissues are quick-frozen in RNA protective solution,then transferred to-80℃ refrigerator spare.2.The immunohistochemical method was used to detect the expression of Kv3.4 protein,EGFR protein and Caveolin-1 protein in each sample,and the result of immunohistochemistry was analyzed to explore the relationship between it and formation of tongue squamous carcinoma.3.The real-time fluorescence quantification PCR method was used to obtain the Δ CT value of Kv3.4,EGFR and Caveolin-1 gene relative to the reference gene GADPH in the tissue samples of each group,thereby to identify the change of the Kv3.4 mRNA,EGFR mRNA,Caveolin-1 mRNA and the specific stage of changes occur during the process of tongue carcinogenesis.Results:1.4NQO water-drinking method was used to successfully induce the occurrence of tongue squamous cell carcinoma of SD rats,4 out of 84 SD rats died during the experiment,a total of 80 experimental samples had been obtained,18 samples for normal group,9 samples for epithelial hyperplasia group,11 samples for epithelial mild dysplasia group,12 samples for moderate epithelial dysplasia group,12 samples for severe epithelial dysplasia group and 18 samples for squamous-cell carcinoma group had been obtained after a pathological grading of lingual mucous membrane tissue of SD rats being collected was done.There is statistical significance of the sample size difference between the pathological grades of tongue mucosal(χ2=44.503,P=0.000,P<0.05).2.the immunohistochemistry test was used to detect the expressions of Kv3.4 protein,EGFR protein,Caveolin-1 protein in all specimens.The positive rate of the three proteins respectively are following.For the expression of Kv3.4,the normal mucosa 44.44%(8/18)is positive epithelial hyperplasia 55.56%(5/9),mild epithlialdysplasia 72.73%(8/11),moderate epithelial dysplasia 75%(9/12)severe epithelial dysplasia 83.33%(10/12)as well as tongue squamous cell carcinoma 94.44%(17/18)are all positive.The expression of Kv3.4 protein is elevated in the process of cancerization of lingual mucous membrane,the difference in expression between different pathological grading is statistically significant(χ2=21.390,P<0.05),comparing two:The positive expression rate of Kv3.4 protein in moderate dysplasia group,severe dysplasia group,tongue squamous cell carcinoma group and normal mucosa group,differences were statistically significant(P<0.05);the differences in positive expression between the remaining groups were not statistically significant(P>0.05).3.For the expression of EGFR,the normal mucosa 38.89%(7/18)is positive epithelial hyperplasia 44.44%(4/9),mild epithelial dysplasia 63.63%(7/11),moderate epithelial dysplasia 66.67%(8/12)severe epithelial dysplasia 75%(9/2)as well as tongue squamous cell carcinoma 88.89%(16/18)are all positive.The expression of EGRF protein is elevated in the process of cancerization of lingual mucous membrane,the difference in expression between different pathological grading is statistically significant(χ2=20.040,P<0.05),comparing two:the difference in positive expression of EGFR protein between the normal lingual mucous membrane group and tongue squamous carcinoma group was statistically significant(P<0.05);the differences in positive expression between the remaining groups were not statistically significant(P>0.05).4.For the expression of Caveolin-1,the normal mucosa 11.11%(2/18)is positive epithelial hyperplasia 22.22%(2/9),mild epithelial dysplasia 27.27%(3/11),moderate epithelial dysplasia 50%(6/12)severe epithelial dysplasia 66.67%(8/12)as well as tongue squamous cell carcinoma 77.78%(14/18)are all positive.The expression of Caveolin-1 protein is elevated in the process of cancerization of lingual mucous membrane,the difference in expression between different pathological grading is statistically significant(χ2=25.946,P<0.05),comparing two:The positive expression rate of Caveolin-1 protein in mild dysplasia group,tongue squamous cell carcinoma group and normal mucosa group,differences were statistically significant(P<0.05);The difference were statistically not significant between the other groups(P>0.05).5.We detected the relative expression levels of Kv3.4 mRNA,EGFR mRNA,Caveolin-1 mRNA in 75 tissue samples by real-time quantitative PCR method.in the 75 tissue samples,there are 17 normal tongue mucosa specimens,9 hyperplasia tongue mucosa specimens,10 mild dysplasia tongue mucosa specimens,11 moderate dysplasia tongue mucosa specimens,10 severe dysplasia tongue mucosa specimens and 18 tongue mucosa cancer specimens.Kv3.4 mRNA averageΔ CT value of normal tongue mucosa group,epithelial hyperplasia group,epithelial mild dysplasia group,epithelial moderate dysplasia group,epithelial severe dysplasia group,epithelial carcinogenesis group is 7.999412,7.617778,7.352000,6.925455,5.780000 and 5.474444.In addition to epithelial hyperplasia group and epithelial mild group,severe group dysplasia and epithelial carcinogenesis group,The difference between the other groups was statistically significant(P<0.05).The vaule of 2-ΔΔ CT of normal lingual mucous membrane group was taken as 1,the value of 2-ΔΔCT of remaining lesion groups was taken as 1.302817,1.566356,2.1052,4.657035 and 5.755604 respectively.Namely,the expression level of Kv3.4 mRNA of the tissue of all lesion groups was respectively 1.302817 times,1.566356 times,2.1052 times,4.657036 times and 5.755607 times as high as that of the tissue of epithelium normal groups.6.EGFR mRNA averageΔ CT value of normal tongue mucosa group,epithelial hyperplasia group,epithelial mild dysplasia group,epithelial moderate dysplasia group epithelial severe dysplasia group,epithelial carcinogens is 6.672400,6.088900,5.661000,4.970000,4.753000 and 4.223900.In addition to epithelial moderate dysplasia group and epithelial severe dysplasia group,the difference between the other groups was statistically significant(P<0.05).The vaule of 2-ΔΔ CT of normal lingual mucous membrane group was taken as 1,the value of 2-ΔΔ CT of remaining lesion groups was taken as 1.49848,2.015866,3.254419,3.782657and 5.458483.respectively.Namely,the expression level of EGFR mRNA of the tissue of all lesion groups was respectively 1.49848 times 2.015866 times,3.254419,3.782657 times and 5.458483 times as high as that of the tissue of epithelium normal group.Caveolin-1 mRNA averageΔ CT value of normal tongue mucosa group,epithelial hyperplasia group,epithelial mild dysplasia group,epithelial moderate dysplasia group,epithelial severe dysplasia group,epithelial carcinogenesis group is 5.565300,5.144400,4.891000,4.760900,4.223000,3.592800.In addition to epithelial hyperplasia group and epithelial mild dysplasia group,epithelial mild dysplasia group and epithelial moderate dysplasia group,the difference between the other groups was statistically significant(P<0.05).The vaule of 2-ΔΔ CT of normal lingual mucous membrane group was taken as 1,the value of 2-ΔΔ CT of remaining lesion groups was taken asl.338762,1.595822,1.746419,2.535552 and 3.924476.respectively.Namely,the expression level of Caveolin-1 mRNA of the tissue of all lesion groups was respectively 1.338762 times,1.595822 times 1.746419 times,2.535552 times and 3.924476 times as high as that of the tissue of epithelium normal group.8.Kv3.4 mRNA,EGFR mRNA,Caveolin-1 mRNA expression m the process of tongue mucosal carcinogenesis,by Pearson correlation analysis showed that the consistency of analysis in the process of tongue mucosal carcinogenesis.Kv3.4 mRNA and EGFR mRNA expression levels were positively correlated(r=0.616,P<0.05);Kv3.4 mRNA and Caveolin-1 mRNA expression levels were positively correlated(r=0.61,P<0.05);EGFR mRNA and Caveolin-1 mRNA showed a positive correlation(r=0.893,P<0.05).Conclusion:1.SD rat tongue carcinogenesis can be induced by 4NQ0 drinking water,however,the man tongue squamous cell carcinoma obtained by this method is highly differentiated squamous cell carcinoma.2.The positive rate of Kv3.4,EGFR and Caveolin-1 were all elevated during the cancenzation of lingual mucous membrane of SD rats,from the dysplasia of lingual mucous membrane to each pathological grading of tongue squamous carcinoma,the expression of Kv3.4 protein,EGFR protein and Caveolin-1 protein were up-regulated with the increase of pathological grade.3.Kv3.4 protein,EGFR protein,Caveolin-1 protein changes at the translational level during SD rat tongue mucosa carcinogenesis,and with the increase of pathological grade,Kv3.4 mRNA,EGFR mRNA,Caveolin-1 mRNA expression showed a increasing trend;In tongue mucosa carcinogenes Kv3.4 mRNA and EGFR mRNA.Kv3.4 mRNA and Caveolin-1 mRNA,EGFR mRAN and Caveolin-1 mRNA expression levels were positively corrrelated.4.Deteccting the expression level of Kv3.4,EGFR and Caveolin-1 in SD rat tongue mucosa carcinogenes is may help to explore the development mechanism of tongue squamous cell carcinoma and provide new ideas or methods about the targeted therapy for tongue mucosa carcinogenesis. |
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