CDC2 Depletion Inhibits The Malignant Progression Of Human Glioma: Experiment In Vitro And In Vivo

Objective: Glioma is the most common and aggressive primary brain cancer. Despite progress in research on the molecular aspects of malignant gliomas, the prognosis of these brain tumors continues to be dismal. One reason for the lack of clinical advances is that the molecular mechanisms of gliomagenesis and progression remain unclear. RNA interference (RNAi) technique is a way by which the 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 CDC2 gene in this research, sequentially as tools to explore the molecule pathogeny and the therapeutic potential of the gene.Methods: The target sequences of siRNA for RNAi of CDC2 gene were designed by the online siRNA selection software in Invitrogen web. 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 and 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 were constructed and transfected into the SHG44-9 glioma cell in vitro. Then the resulting phenotypic changes of target gene were investigated as follows. The level of CDC2 mRNA was measured utilizing Real-time PCR; the CDC2 protein level was subject to Western blotting; the cell cycles and apoptosis were detected by using flow cytometry. And the viral particles containing small interfering RNA for CDC2 were subsequently injected into xenogeneic graft tumor of nude mice subcutaneously and intracranially. To evaluate the therapeutic potential of CDC2 deplition xenogeneic graft tumor volume and survival of nude mice were measured.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 titer of the pSUPER.retro-C3 was 4.25×105CFU/ml. Human glioma cells SHG-44 (overexpression of CDC2) transfected with retrovirus vector expressing shRNAs targeting CDC2 were arrested in G2/M phase. Proliferation of tumor cells was suppressed by 92% 48h after transfection and apoptosis was increased from 3.97% to 36.52%. Weight of human glioma xenografts in nude mice treated with saline as control was 102.6±75.2 mg, and those treated with recombinant viral particles was 32.0±16.5 mg (P=0.021). Compared with animals treated with PBS, a significant increase in survival occurs in those treated with recombinant C3 retrovirus (p<0.05).Conclusion: Successful construction of pSUPER recombinant retrovirus vectors expressing siRNA for CDC2 gene knockdown supplies useful tools for studying the molecule pathogeny and new gene therapy of gliomas sequentially, and provides a new worktable for researching other tumors overexpressing CDC2 gene as well. CDC2 gene plays an important role in the maintenance of proliferation of human gliomas. CDC2 plays an important role in the maintenance of proliferation of human gliomas. Downregulation of CDC2 could prevent the progression of the malignant phenotype of human gliomas. And CDC2 could become a potential target on gene therapy of human gliomas.