The Preliminary Research For The Effect And Molecular Mechanism Of PRDX-1in Malignant Progress Of Glioma

PurposeGlioma is common in intracranial malignant tumors，the traditional surgery andradiation chemotherapy treatments do not get significant effect.Researchers found that afterdifferention therapy with Nordy，not only the proliferative activity of glioma cell U87MG isdecreased，the level of intrastitial PRDX-1(Peroxiredoxin1) is also significantly reduced，which presents that PRDX-1may be closely related to the malignant change of glioma.Wedesigned the experimens to measure the expression of PRDX-1，PTEN，EGFR，PI3K，Nrf2，NF-κB，TNF-α in human glioma tissue to detecte the relationship between thoseproteins and degrees of malignant glioma tissue.Constructing the lentiviral expressionvector and lentiviral inhibit expression vector，transfect them into Glioma cell line U87MG，then measure the changes of cell biology characteristics，investigate the mechanism ofPRDX-1in the process of glioma malignant transformation.MethodsThe glioma specimens of pathology department in Xinqiao Houspital from2010to2011were searched and graded according to the WHO2007standards.The expressions ofPRDX-1，PTEN，EGFR，PI3K，Nrf2，NF-κB，TNF-α of glioma were examed withimmunohistochemical technique， the electronic images taken by microscope werequantitative analysised in form of Mean Optical Density(MOD) which is counted by ImagePro Plus6.0.Three Groups of short hairpin RNA(shRNA) lentiviral vectors targeting humanPRDX-1were constructed and transfected into human glioma cell line U87MG.The level ofPRDX-1mRNA were detected by Real-time polymerase chain reaction，and the level ofPRDX-1protein were detected by Western blot，then selected the cell line which got the themost inhibitory effective targeting human PRDX-1.Human PRDX-1gene coding regionwas cloned into lentiviral vector，then transfected into human glioma cell line U87MG.Thelevel of PRDX-1mRNA were detected by Real-time polymerase chain reaction，and the level of PRDX-1protein were detected by Western blot.All cell lines were determined byflow cytometry，CCK-8assay，colony assay to detected the change of proliferation，apoptosis and colony forming ability.Immunocytochemistry was also be taken to detectedthe expressions of PRDX-1，PTEN，EGFR，PI3K，Nrf2，NF-κB，TNF-α in those celllines.All data were analyzed by SPSS19，comparison between multiple groups of averagewere analyzed by one-way analysis of variance and Dennett’s T3test，comparison betweentwo groups of the average were analyzed by T-Test.ResultsThe expressions of PRDX-1，EGFR，PI3K，Nrf2，NF-κB，TNF-α in high-grade gliomaare significantly higher than those in low-grade glioma(P＜0.05)，and show significantupward trend with glioma grades synchronously，and PTEN presents the opposite result(P＜0.05).Three Groups of pHBLV-PRDX-1-shRNA lentiviral vectors targeting humanPRDX-1gene and their blank control entiviral vector have been successfully constructedand maintains high expression in U87MG cells.The cell line which got the most inhibitoryeffective targeting human PRDX-1was selected according to resluts of RT-PCR andWestern blot.Compared with U87MG before transfection and U87MG transfected withblank control entiviral vector，the one transfected with pHBLV-PRDX-1-shRNA lentiviralvector get longer and more protuberances appearance，and has less mitotics.The expressionsof PRDX-1both in mRNA level and protein level are reduced，apoptosis is increased，cellgrowth and colony forming activity are restricted.The PRDX-1，NF-κB，TNF-α expressionlevels which were detected by immunocytochemistry show significantly reduce，in contrastthe expression levels of PTEN， PI3K， Nrf2， EGFR do not have significantchange.Compared with U87MG before transfection and U87MG transfected with blankcontrol entiviral vector，those transfected with pHBLV-PRDX-1lentiviral vector get shorterand less protuberances appearance.The expressions of PRDX-1both in mRNA level andprotein level are increased，apoptosis reduceded，cell growth and colony forming activityare promoted.The PRDX-1， NF-κB， TNF-α expression levels detected byimmunocytochemistry show significantly rise，in contrast the expression levels of PTEN，PI3K，Nrf2，EGFR do not have significant change.ConclusionsThe expression level of PRDX-1in hunman glioma tissues is closely associated with glioma grades，it also show significant influence to proliferation，apoptosis and cloneformation.The PRDX-1which is amplified by Nrf2inside glioma cells can act on TNF-α tosend functional NF-κB，involves in the regulation of nuclear factor and lead to the reductionin apoptosis level and promote malignant transformation of glioma cells.PRDX-1can alsoinfluence EGFR and its downstream PI3K/AKT pathways by forming complexes withPTEN，which may participate in the process of malignant transformation in glioma cells，but the mechanism still needs a further study.