Dominant-Negative Of Survivin's C-flanking Sequences For The Therapy Of Colon Cancer Cell Line

Backgroud/ Objective:Colorectal carcinoma(CRC)is a kind of malignant tumor which has high attack rate and death rate.Biotherapy such as gene therapy and immunotherapy is the tendency to cure the disease of the further.The success of cancer gene therapy and immunotherapy should be the tools which can kill the cancer cells as much as possible meanwhile keep the normal cells in safety.The success of cancer immunotherapy is dependent on using suitable tumor-associated antigens (TAAs).An ideal TAA should have several important features.1)TAA should induce T cells that recognize tumors but not normal cells.2)TAA should be expressed in tumors from a significant proportion of the patients and in a significant proportion of tumor cells.3)It is important that TAA belongs to molecules,which are obligatory for the survival of tumor cells.Immunization against such TAA(s)would overcome tumor escape attributable to antigenic variation because losing the survival-related molecule may result in tumor cell death.At this time,there are very few molecules that fit these criteria.Survivin may be one such rare candidate.First,Survivin is expressed at a high level in virtually every human cancer.Second,Immune responses to Survivin include the generation of antibodies and type 1 T-helper cells and CTL responses in cancer patients.Thirdly,It also plays a critical role in the survival of cancer cells.The attractiveness of Survivin make it as a excellent target of anticancer gene therapy and immunotherapy.Most of the immunocompetence polypeptide is in the region of the C-flanking sequences of Survivin and the key site 84^thamino acids (half-cystine)is in this region too.When the 84^thhalf-cystine(Cys)of Survivin gene was mutated to alanine(Ala)(C84A),the endogenous Survivin will displace and lead to "Mitotic Catastrophe" and apoptosis.In this case C-flanking sequences of Survivin does has the ability to to embody the main function and immunogenicity of full length wild Survivin and should be the potential target for tumor therapy.In this study, we will clone the C- flanking sequence of Survivin and construct a Eukaryotic Expression Vector of C-terminal of Survivin.Make mutation of the 84^thAmino Acid from Cys to Ala to construct the Eukaryotic Expression Vector of C-terminal of Survivin with the Dominant-Negative Mutant on the 84^thAA.Then make a evaluation of these new constructed vector for their function in treatment of colon cancer cell line.Methods:1)C-flanking sequences of Survivin gene was amplified from pCDNA3.1-sur by PCR and inserted into pEGFP-C1 to generate a recombinant construct vector:pEGFP-C1-Csur.The recombinant plasmid was detected by PCR and double digestion by both HindⅢand BamH.Sequencing was done subsequencely and the result was compared to the database of GeneBank.2) Method of directly mutation was used to mutate the 84th Amino Acid in the pEGFP-C1-Csur and pCDNA3.1-Msur/T34A from Cys to Ala.Eukaryotic Expression Vector of pEGFP-C1-MCsur/C84A and pCDNA3.1-Msur/T34A-C84A were Constructed.Sequencing was done and the result was compared to the database of GeneBank subsequencely.3)Methods of RT-PCR and Western-Blot was used to demonstrate the expression of the recombinant vector after its transient transfection into lovo cell line(a kind of colorectal cancer cell line).4)Fluorescence microscope was used to detect the apoptosis of the transient-transfected Lovo cell line with different recombinant plasmid.The Lovo cell was dyed by Hoechst 33258.5) Flow cytometer(FCM)was used to detect the cell cycle of the transient-transfected Lovo cell line with different recombinant plasmid.The Lovo cell was dyed by Propidium Iodide(PI).Result:1)C-flanking sequences of Survivin gene was amplified and inserted into pEGFP-C1 successfully subsequenct to the detection of PCR,double digestion by both HindⅢand BamH.The homology between sequencing result and that of Survivin gene CDS presented in GeneBank was 100%.2)The result of sequence suggested that the mutation in the recombinant construct vector: pEGFP-C1-MCsur/C84A and pCDNA3.1-Msur/T34A were successfully.3)The result of RT-PCR and Western-Blot test showed the expression of mRNA and protein were the exactly the same as prediction.Group of Lovo(untreated)and pCDNA3.1(+)showed lower concentration of Survivin protein with molecular weight of about 16.5kd meanwhile all the recombinant construct vector treated group of pCDNA3.1 -sur,pEGFP-C1-Csur,pEGFP-C1-MCsur/C84A,pCDNA3.1-Msur/T34A -C84A showed a much higher concentration of Survivin protein.Comparion to other groups suggestd that group of pEGFP-C1-Csur and pEGFP-C1-MCsur/C84A show a confluence protein(EGFP/ C-flanking of Survivin protein)with molecular weight of about 31kd.The relative expression of mRNA(The relative expression of mRNA=Survivin gray scale value/GAPDH gray scale value)in groups of Lovo(untreated),pCDNA3.1(+),pCDNA3.1-sur,pEGFP-C1-Csur,pEGFP-C1-MCsur/C84A,pCDNA3.1-Msur/T34A-C84A are 0.246±0.023,0.263±0.011,0.454±0.058,0.627±0.028,0.736±0.137,0.450±0.054 respectively. The relative expression of mRNA in groups of Lovo(untreated)and pCDNA3.1(+) are lower than other 4 group obviously(P= 0.00).4)Apoptosis was detected in Lovo cell(dyed by Hoechst 33258)obviously aider transient-transfected by pEGFP-C1-MCsur/C84A and pCDNA3.1-Msur/T34A-C84A.The same situation was not observed in groups of pCDNA3.1(+),pCDNA3.1-sur and pEGFP-C1-Csur.5)The Gl%of Lovo cells(dyed by PI)in different groups are (46.34±3.67)%(Lovo),(42.38±1.60)%(pCDNA3.1(+)),(47.75±1.79)% (pCDNA3.1-sur),(48.81±4.60)%(pEGFP-C1-Csur),(54.52±2.62) %(pEGFP-C1-MCsur/C84A),(60.524±1.30)%(pCDNA3.1-Msur/T34A-C84A). The Love cell were block in cell cycle Gl in groups of pEGFP-C1-MCsur/C84A and pCDNA3.1-Msur/T34A-C84A(P=0.00),and the situation in group of double mutation are more conspicuous,P=0.024.The same situation was not observed in group pCDNA3.1(+),pCDNA3.1-sur and pEGFP-C1-Csur.Conclusion:1)The construction and expression of Eukaryotic Expression Vector of pEGFP-C1-Csur,pEGFP-C1-MCsur/C84A,pCDNA3.1-Msur/T34A-C84A were successfully by using the techniques of genetic engineering and rite-directed mutagenesis techniques.2)C84A in either C-flanking sequences of Survivin gene(pEGFP-C1-MCsur/C84A)or full length Survivin gene(pCDNA3.1-Msur/T34A -C84A)will result in the antagonism of the inhibition apoptosis function of wild form Survivin.The same situation was not observed in group of pEGFP-C1-Csur which suggested that the Survivin gene(lack of N- flanking)should be able to induce apoptosis inhibition and this apoptosis inhibition function can be depressed by C84A.3)C84A in either C-flanking sequences of Survivin gene or full length Survivin gene will result in the block of cell cycle in Gl which will cause problems in times of cell mitosis.Cell proliferation will decreased subsequencely.In a word,theC-flanking sequences of Survivin does has the ability to to embody the main biology function of full length wild Survivin and use the method of directly mutation to mutate the 84th Amino Acid from Cys to Ala will result in the apoptosis and block of cell cycle in Gl.