Construction Of ShRNA-expressing Plasmid Targeting EZH2and Its Effects On The Apoptosis And Proliferation Of Human Brain Glioma U251Cells

Background:Glioma are the most frequent malignant tumors of the central nervous systemand accounting for approximately45%of all intracranial tumors. The currenttreatment measures of glioma include intracranial surgery, radiotherapy,chemotherapy, and comprehensive treatment. Despite the recent great advances insuch treatment measures, the overall outcome of patients with malignant glioma wasstill poor due to the soaring proliferation and invasivenvess of glioma cells.Nowadays, gene therapy was regarded as a promising approach for glioma treatment,but there is still a lack of ideal and reliable therapeutic target. So it has become one ofthe focus of neurosurgery to find the molecular target which regulated glioma cells’malignant behavior and clarify the pathological mechanisms in glioma pathogenesis.Polycomb protein EZH2is the catalytic subunit of Polycomb repressive complex2(PRC2), which is a highly conserved histone methyltransferase that targets lysine-27of histone H3. This methylated H3-K27chromatin mark is commonly associated withsilencing of differentiation genes in organisms ranging from plants to flies to humans.Studies on human tumors show that EZH2is frequently over-expressed in a widevariety of cancerous tissue types, including prostate,breast and glioma. Its highexpression can promot tumor cell proliferation, invasion and metastasis viasupressing a number tumor suppressor gene expression.Therefore EZH2wasconsidered to be a new candidate oncogene. RNA interference is a kind of genesilencing technology occurred and developed rapidly in recent years. It can degradetarget gene mRNAspecially by importing specific fragments of double-stranded RNA molecules homohogous to the target gene into cells. It is g a powerful tool for genediagnosis and gene therapy.ObjectiveTo construct and screen shRNA-expressing plasimd targeting to EZH2gene.To study the effects of EZH2gene’s inhibition by RNA interference on theproliferation and apoptosis of human glioma U251cells.MethodsThe DNA oligonucleotide fragments targeting to human EZH2gene weredesigned and synthesized,then were cloned into pGPU6/GFP/Neo plasmid.Therecombinant plasmids were identified by restriction enzyme and sequencinganalyses,then were transfected into U251glioma cells; the transfection efficiencywas observed and the EZH2gene silencing effect was detected by quantitativeRT-PCR and Western bloting. The shRNA-expressing plasmid targeting EZH2gene was constructed and transfected into U251cells.Qrt-PCR and Western blot were used to detect the EZH2gene’s exprsssion atthe level of mRNA and protein；MTT assay was performed for detecting cellproliferation and Annexin V-FITC/PI flow cytometric analysis for cell apoptosis.ResultsThe recombinant plasmids were constructed and transfected into U251gliomacells successfully, the transfection rate was approximately70%. Of which,theinhibition efficiency of the reconbinant plasimd targeting hEZH2-715sequencewas the best. It’s inhibition rates were55%and89%in the level of EZH2mRNA and protein in the U251cells respectively.After the transfection of the plasmid, the growth of U251cells was signifinantlyinhibited (p<0.001), and the highest growth inhibition rate was35.79%in96 hours. The early and secondary apoptosis rate of U251cells were26.59%and38.63%respectively, which were higher than both the blank control group andthe negative control group (P<0.01).Conclusions：The recombinant plasimd expressing EZH2-shRNA that could suppress EZH2gene’s expression effectively was constructed successfully, which laid thefoundation for next step to explore EZH2gene’s the biological role in gliomacells.The silence of EZH2gene in glioma U251cell could inhibit itsproliferation, promote its apoptosis, suggesting that EZH2may become a newtarget for glioma gene therapy.