The Experimental Study On RNAi Of The Genes Related To Occurrence And Progression Of Human Brain Glioma Cells In Vitro

Objective: Survivin gene and cdc2 gene are related to malignant progression of gliomas. But the effects of the two genes in the occurrence and development of glioma cells are still obscure. RNA interference (RNA interference, RNAi) technique is a way by which the double-stranded RNA (double-stranded RNA, dsRNA) intermediates the degradation of its homological mRNA of given gene and the specific inhibition of the gene expression subsequently in cells. To design and construct the recombinant retrovirus vectors expressing siRNA for RNAi of survivin , cdc2 gene in this research, sequentially as tools to explore the function of the two genes,then study the biological behavior changes of the low-differentiated SHG44-9 glioma cell strain with RNAi of survivin gene for exploring the molecule pathogeny and new gene therapy of gliomas.Methods: The target sequences of siRNA for RNAi of cdc2 gene were designed by the online siRNA Selection Program and siDirect software. The target sequences of siRNAs for RNAi of survivin gene were designed by siDirect. The DNA sequences containing a palindrome structure were synthesized and annealed to form double-stands which were inserted into linear pSUPER plasmid vectors later. The amplified and purified recombinant plasmids were identified by agarose gel electrophoresis after cleaving of double enzymes and by sequencing, then were transfected into the phoenix cells, which produced retrovirus. The titers of the recombinant retroviruses were determined by NIH3T3 cell. The recombinant retrovirus vectors expressing siRNA for RNAi of cdc2 gene,the pSUPER.retro-Cl and pSUPER.retro-C2,were constructed. Then the pSUPER.retro-S 1 and the pSUPER.retro-S2 retroviruses, the retrovirus vectors expressing siRNA for knockdown of survivin gene, were constructed to transfect the SHG44-9 glioma cell strain. The expression level of survivin in transfected SHG44-9 glioma cells was detected by western blot and RT-PCR. The cell cycle and cell proliferation was analyzed by flow cytometry and MTT respectively while the apoptosis of target cells change was detected by TUNEL assay.Results: The recombinant pSUPER plasmid vectors expressing siRNA were confirmed by agarose gel electrophoresis after cleaving of double enzymes and by sequence analysis. The inserted 60bp sequences were identical to the original sequences and in corresponding position. The titers of the pSUPER.retro-Cl and pSUPER.retro-C2 were 4.25xlO5CFU/ml and 6*105 CFU/ml respectively. The titers of the pSUPER.retro-Sl and pSUPER.retro-S2 were 5.5xlO5CFU/ml and 5.75*105 CFU/ml respectively. .The survivin inhibitory effect of pSUPER.retro-Sl in SHG44-9 glioma cell was 68.03±0.02% and 70.5% by RT-PCR and western blot analysis respectively while the inhibitory effect of pSUPER.retro-S2 was 89.90±0.02% and almost 100% respectively. The SHG44-9 cells with knockdown of survivin gene displayed following changes: arrest in G2/M phase of the cell cycle ; inhibition of the cell proliferation; increase of apoptosis.Conclusion: Successful construction of pSUPER recombinant retrovirus vectors expressing siRNA for survivim cdc2 gene knockdown supplies useful tools for studying the molecule pathogeny and new gene therapy of gliomas sequentially, and provides a new worktabie for researching other tumors overexpressing survivin .. cdc2 gene as well. Growth of the SHG44-9 cells with RNA interference of survivin was inhibited with increase of apoptosis at the same time .Survivin gene can be applied as a new target of gene therapy of gliomas. The recombinant retrovirus vectors expressing siRNA targetingsurvivin gene had extended effect of RNA interference,which will be applied to the research of treating the animals burdened with human brain glioma in vivo.The functions of cdc2 gene will be determined by the continued study.