| Backgrounds:Glioma is the most common intracranial tumors with highly aggressive and invasive.Because of the unclear boundary,removing it completely by a one-off surgery is difficult.Epithelial-mesenchymal transition(EMT)and mesenchymal-to-epithelial transition(MET)are reversal progress,which correlate with the tumorigenesis and tumor progression.EMT has a potential to promote the distant metastasis and ectopic invasion,while MET promote the proliferation and differentiation of tumor cells.Resveratrol has a potential to resistant to tumors as a natural polyphenol compounds and a certain anti-tumor effect to gliomas.The proteomics data analysis in our lab showed that the expression of Keratin 17 is obviously reduced.KRT 17 was identified as a marker of epithelial tumor,which reminders us that resveratrol probably down-regulate the abnormally high expression of epithelial markers.Therefore,this study aims to prove the existence of the tendency of mesenchymal-epithelial transformation,and to detect and analyze the adjustment of resveratrol in glioblastoma cell lines on the mesenchymal-epithelial transformation.There are some concrete research contents as follow:1.Detect the expression of KRT 17,E-cadherin,N-cadherin and beta-catenin in glioma tissues and analyz the correlation with tumor classification;2.Analyze the correlation of KRT 17 with E-cadherin;3.Determine the sensitivity of glioblastoma cell lines LN428,LN18 and U251 towards resveratrol;4.Compare the expressions of KRT 17,E-cadherin,N-cadherin and beta-catenin in LN428,LN18 and U251 before and after resveratrol treatment;5.Detcet the nuclear translocation of beta-catenin in LN428,LN18 and U251,and analyze the effect of resveratrol on it;6.Prove the tendency of mesenchymal-epithelial transformation in glioma,and discover the reversal effect of resveratrol on the transformation.Methods:By combination of paraffin embedded tissue array and immunohistochemistry(IHC),the expression of KRT 17,E-cadherin,N-cadherin and beta-catenin in 95 cases of human glioma tissues were detected.The data of their expressions were statistically analyzed with tumor classification by SPSS 18.0 software,and analyze the correlation of expression of KRT 17 with E-cadherin and beta-catenin by Spearman Rank and Bivariate Correlation.H-E staining and MTT analysis were used to determine the sensitivity of glioblastoma cell lines LN428,LN18 and U251 towards resveratrol.RT-PCR,ICC and Western-blotting were used to detect the expression changes of KRT17,E-cadherin,N-cadherin and beta-catenin before and after the treatment of resveratrol.IF was used to detect the nuclear translocation of beta-catenin in LN428,LN18 and U251 and to analyze the effect of resveratrol on it.Results:1.The expression of KRT 17,E-cadherin,N-cadherin and beta-catenin in IHC test of glioma arrays:(1)The comparison of the positive rate: the positive rate of KRT 17,E-cadherin,N-cadherin and beta-catenin in gliomas are 67.8%,27.8%,100% and 91.9%,while0%,20%,100%,60% in peritumoral tissue.And the positive rate of KRT 17 and beta-catenin were higher than peritumoral tissue.(2)The analysis of correlation with tumor classification: beta-catenin is positively correlate and the date are not statistically significant(p=0.045,r=0.212).KRT 17(p=0.493,r=0.028),E-cadherin(p=0.474,r=-0.017),N-cadherin(p=0.499,r=0.014)have no correlation with tumor classification.(3)The expression of KRT 17 has positive correlation with E-cadherin(p=0.023,r=0.232)while the difference is not significant.2.Determine the sensitivity towards resveratrol: the results of H-E staining and MTT method showed that after treatment of resveratrol for 48 hours the number of LN428 cells remains the same and cell morphology unchanged demonstrating the chemoresistance,LN18 changes a less and resistant slightly towards resveratrol.The number of U251 cells reduced a lot and morphology changed obviously performing its sensitivity.3.The detection of the relevant biological molecules with MET before and after resveratrol treatment:(1)KRT 17 as cytoskeleton protein expressed positively in LN428 and reduced obviously after treatment,while in LN18 and U251 expresed negatively.(2)E-cadherin as epithelia maker expressed positively and down-regulated in LN428,and extently expressed in LN18 by PCR,while ICC and western showed negatively,slightly expressed and negatively expressed in U251.(3)N-cadherin as mesenchymal maker is positively expressed in LN428,LN18 and U251.The expression of N-cadherin is down-regulated in LN428,unchanged in LN18.PCR showed no difference while ICC and western showed down-regulation in U251.(4)beta-catenin is positively expressed in LN428 and PCR showed invariance while ICC and Western-blot showed slight decrease.It is negatively expressed in LN18 and U251.4.IF test demorstrated the nuclear translocation of beta-catenin occured in LN428 and resveratrol down-regulated the expression of beta-catenin in nuclear.Conclusion:1.The positive rate of KRT 17 and E-cadherin in glioma tissues are 67.8% and 27.8%,which are higher than 0% and 20% in peritumoral tissue.The up-regulation of epithelial maker indicated the tendency of mesenchymal-epithelial transformation.2.The expression of beta-catenin has positive correlation with glioma classificationwhile the difference is not significant(p=0.045,r=0.212).The expression of KRT17 has positive correlation with E-cadherin(p=0.023,r=0.232).The results indicated that with the higher tumor malignant degree,the invasive and infiltrative ability is stronger.3.After the treatment of resveratrol,the expression of KRT 17 and E-cadherin are down-regulated showing the epithelial potential is weakened.The expression of N-cadherin and beta-catenin in nuclear are down-regulated showing that resveratrol has potential to reverse the process of mesenchymal-epithelial transformation and to inducing tumor cell apoptosis and exert anti-cancer effect.4.After the treatment of resveratrol,the expression of epithelial maker in glioblastoma cells having different drugs sensitivity is down-regulated,which indicated that resveratrol can induce the cells resistant towards itself to regain the mesenchymal potential of gliocyte.5.Speculate that there was tendency of mesenchymal-epithelial transformation and demonstrate that resveratrol was able to reverse this process and promote the tumor cells to differentiate to mesenchymal cells. |
PDF Full Text Request