Study Of Regulating Effect Of Transcriptional Factor E2F On The Op18 Gene Expression And Tumor Phaenotype Reversing

Op18 (Oncoprotein 18, Stathmin), a signal transduction regulatory factor in both healthy cells and malignant cells, plays a crucial role in cell division and malignant tumor development . Op18 is highly expressed in many cancers but not in normal cells . Op18 has been discovered and identified to be a very important biotherapy target in the tumor gene therapy. However, the transcriptional mechanisms responsible for its normal regulation and for its high level over expression in malignant cells remain poorly understood. Op18 gene is a direct target of transcription factor E2F . Transcription factor E2F is implicated in cell cycle regulation and is important in cellular transformation.In this study,we attempt to explore transcriptional mechanisms that Op18 is mediated through transcription factor E2F for a new strategy to cancer gene therapy .Objective:To investigate the blocking effect of E2F decoy oligodeoxynucleotides (E2Fdecoy ODNs) in dumbbell shape on op18 gene transcription and the tumor cells proliferation; To construct E2F1 siRNA retroviral expression vector driven by tumor specific survivin promoter and explore its specific and targeted gene therapy by down-regulating op18 expression and decreasing tumor cells proliferation. Methods:(1)E2F decoy ODNs were designed and synthesized,and then transfected into tumor cells(MKN-45 cells and A549 cells)using lipofectamineTM2000 reagent according to the manufacturer's instructions.The level of op18 mRNA in transfected tumor cells was detected by RT-PCR.The proliferation of transfected tumor cells was analyzed by MTT assay and the apoptosis of the tumor cells was detected by TUNEL staining.(2)Survivin promoter and E2F1 siRNA sequences were subcloned into retroviral vector pLXSN by recombinant DNA technique. Retroviral vector pLXSN/Surp/E1 was packaged with PA317 cells and selected in G418 to obtain the positive clones, which were able to produce stable recombinant retrovirus.A549 cells were transduced with the recombinant retrovirus.The positive clones were obtained after G418 selection and were termed A549/E1.The integration and expression of neo gene on parental vector in A549/E1 cells were identified by PCR.(3)The level of op18 expression in A549/E1 cells was detected by RT-PCR and Western blot methods.(4)The effects of recombinant retroviral vector for proliferation on A549/E1 cells were assessed by MTT.Finally the FCM method were used to analyze cell apoptosis.Results: (1) E2F decoy ODNs could be successfully transfected into tumor cells and RT-PCR results indicated that E2F decoy ODNs decreased expression of op18 gene in transfected tumor cells(MKN-45 cells and A549 cells).The proliferation of tumor cells was inhibited markedly by E2F decoy ODNs and morphologic analysis showed positive apoptosis staining identified by TUNEL assay.(2) By sequencing, restriction digestion and PCR ,we confirmed that the sequence, length, position and orientation of inserted genes were all correct. Then the recombinant retroviral vector pLXSN/Surp/E1 was constructed successfully. The retroviral vector pLXSN/Surp/E1 was packaged with PA317 cells and stable titer producing cell C28 was established(2.0×106/ml). PCR results demonstrated that survivin promoter and E2F1 siRNA gene were integrated into the A549/E1 cells genome after A549 cells were transduced with the recombinant retrovirus. RT-PCR results indicated that E2F1 siRNA decreased expression of op18 gene in A549/E1 cells.The level of op18 protein expression in A549/E1 cells was down-regulated by E2F1 siRNA and the proliferation of A549/E1 cells was inhibited markedly. The FCM results showed cell apoptosis increased in A549/E1 cells.Conclusion: E2F decoy ODNs may specifically decrease the activity of transcription factor E2F,and further down-regulate op18 gene expression and inhibit tumor cells proliferation; The survivin promoter-driven E2F1 siRNA for gene silencing is specific to tumor cells; Suppression of E2F1 by using RNAi-targeted system not only could strongly inhibit cell proliferation, but also could significantly decrease expression of op18 gene. Our study provides experimental foundation for targeted gene therapy on E2F and op18 gene.