Experimental Studys On The Expression Of PRDXâ…¢ In Human Gliomas And PRDXâ…¢ Gene Silence Inducing Apoptosis By RNAi In U251 Cells

Objective: The purpose of this study was to investigate the expressionlevel of PRDXⅢin different grade of gliomas and effect on apoptosis.Methods: The expression level of PRDXⅢprotein in 65 gliomas and10 normal brain tissue samples was detected by immunohistochemistry andthe apoptotic index of cells was inspected by Tunnel. The expression level ofPRDXⅢmRNA in 29 gliomas and 9 normal brain tissue was detected byRT-PCR. We analyzed the difference of expression of PRDXⅢin differentgrade of gliomas and normal brain tissues and the relationship between thegrades of gliomas and apoptotic index. The specific PRDXⅢsiRNAs weredesigned and transfected into U251 cells by liposome. The expression levelof PRDXⅢmRNA and protein were detected by RT-PCR and Westblottingrespectively after transfection to select effective siRNA. The apoptoticsituation was detected by flow cytometer (FCM), DNA ladder and PI stainingand the cellular growth inhibition ratio by MTT 48 hours after transfection.Results: PRDXⅢprotein was expressed in 2 of 10 (20%) normalbrain tissues, 4 of 14(28.6%) gradeⅠgliomas, 6 of 19(31.6%) gradeⅡgliomas, 10 of 13(76.9%) gradeⅢgliomas and 16 of 19(84.2%) gradeⅣgliomas. The difference of normal brain tissues, gradeⅠgliomas and gradeⅡgliomas was not significant, neither was the diference between gradeⅢgliomas and f gradeⅣgliomas. The expression level of PRDXⅢingradeⅢgliomas and gradeⅣgliomas was significantly higher than in normal brain tissues, gradeⅠand gradeⅡgliomas. By spearman correlationanalysis, acompaning the increasing of glioma grades, the expressingintensity of PRDXⅢincreased significantly too (r=0.576,p=0.00). Thecellular apoptotic index was 24.51±3.27％,18.90±2.69％,16.46±2.74％,9.90±3.17％和7.66±2.83％in normal brain tissues, gradeⅠgliomas,gradeⅡgliomas, gradeⅢgliomas and gradeⅣgliomas respectively.Following the increasing in tumor grades, the apoptotic indexs graduallydegraded (F=74.45,P=0.00). The apoptotic index was 10.29±4.87％innormal brain tissues and gliomas expressed PRDXⅢand 19.07±4.64％inthose did not expressed PRDXⅢ. The difference was significant. The ratio ofPRDXⅢmRNA OD value andβ-actin OD value was 0.48±0.12, 0.69±0.22,0.96±0.24 and 0.97±0.36 in normal brain tissues, gradeⅡgliomas, gradeⅢgliomas and gradeⅣgliomas respectively. Following the increasing ofglioma grade, the ratios gradually increased too (F=8.17, P=0.00). Theexpression of PRDXⅢmRNA and protein were reduced significantly aftertrnsfection in interference-1 group and interference-3 group by RT-PCR andwestern-blotting. The inhibitory action was more effective in interference-3group than interference-1 group. The expression of PRDXⅢmRNA andprotein were reduced by 76％and 63.9％respectively in interference-3group. DNA ladder showed obvious ladder-shaped electrophoresis strip ininterference group, but no ladder-shaped electrophoresis strip in all the othergroups. PI staining showed that some cellular nucleus were stained thicklyand some were splited into bits in interference group, which was consistantwith apoptotic morphological changes. FCM showed that the cellularapoptotic rates were 2.26±0.64％, 2.68±0.74％, 3.54±0.62％and 35.9±4.85％in blank control group, mock vector group, unconcerned siRNA group and interference group respectively. The apoptotic rate was more obvious ininterference group than in all the control groups (P=0.00).MTT showed thatthe growth of U251 cells was inhibited by 44.4％.Conclusions PRDXⅢwas expressed in glioma tissues and theexpressing intensity increased when the glioma grade went up. Theexpression of PRDXⅢwas more intensive in gradeⅢand gradeⅣgliomasthan in normal brain tissues, gradeⅠand gradeⅡgliomas,which indicatedmaybe PRDXⅢwas related to the growth and malignant phenotype ofgliomas.The cellular apoptotic indexes were much smaller in gliomasexpressed PRDXⅢthan in those did not express PRDXⅢ. The PRDXⅢmRNA and protein of U251 cells were inhibited by RNAi and cell apoptosiswas induced successfully, which indicated PRDXⅢwas related to gliomaapoptosis and PRDXⅢgene may become one of the target gene in genetherapy of gliomas.