Study On The Biological Effect Of Multiple SiRNA Recombinant Expression Vector Targeting Livin And Survivin Gene Simultaneously To Human Colon Cancer Cells

Part 1: Study On The Biological Effect Of Combining SiRNARecombinant Expression Vector Targeting Livin Gene With5-Fu To Human Colon Cancer CellsSummary Objective To construct SiRNA recombinant expression vector targetingLivin gene and observe the apoptosis induction effect of it in human colon cancer cells, andresearch the enhancement of chemotherapy sensitivity. Methods SiRNA recombinantexpression vector targeting Livin gene was constructed and transfected into human coloncancer cell.The effect of SiRNA recombinant expression vector was detected by RT-PCR,Western blot, MTT reduction assay and flow cytometry. Enhancement of chemotherapysensitivity was observed by MTT reduction assay and flow cytometry. Results It wasconfirmed by restriction endonuclease and sequence analysis that SiRNA recombinantexpression vector targeting Livin gene was constructed successfully. Inhibition ratio ofLivin SiRNA at mRNA and protein levels were 30.18% and 28.88%. The growth ofcancer cells was inhibited significantly and the apoptotic ratio was 13.36±1.45 %. It couldenhance chemotherapy sensitivity by combining 5-Fu. Conclusions The SiRNArecombinant expression vector targeting Livin gene has been constructed successfully. Itnot only can inhibite the expression of Livin gene but also can induce apoptosis in humancolon cancer cells remarkably. And it can enhance chemotherapy sensitivity and apoptosisinduction effect by combining 5-Fu in human colon cancer cells significantly. Part 2: Study On The Biological Effect Of Combining SiRNARecombinant Expression Vector Targeting Survivin GeneWith 5-Fu To Human Colon Cancer CellsSummary Objective To construct SiRNA recombinant expression vector targetingSurvivin gene and observe the apoptosis induction effect of it in human colon cancer cells,and research the enhancement of chemotherapy sensitivity. Methods SiRNA recombinantexpression vector targeting Survivin gene was constructed and transfected into humancolon cancer cell.The effect of SiRNA recombinant expression vector was detected byRT-PCR, Western blot, MTT reduction assay and flow cytometry. Enhancement ofchemotherapy sensitivity was observed by MTT reduction assay and flow cytometry.Results It was confirmed by restriction endonuclease and sequence analysis that SiRNArecombinant expression vector targeting Survivin gene was constructed successfully.Inhibition ratio of Survivin SiRNA at mRNA and protein levels were 36.33 % and 44.65%.The growth of cancer cells was inhibited significantly and the apoptotic ratio was17.24±2.13 %. It could enhance chemotherapy sensitivity by combining 5-Fu. ConclusionsThe SiRNA recombinant expression vector targeting Survivin gene has been constructedsuccessfully. It not only can inhibite the expression of Survivin gene but also can induceapoptosis in human colon cancer cells remarkably. And it can enhance chemotherapysensitivity and apoptosis induction effect by combining 5-Fu in human colon cancer cellssignificantly. Part 3: Study On The Biological Effect Of Multiple SiRNA RecombinantExpression Vector Targeting Livin And Survivin GeneSimultaneously In Human Colon Cancer CellsSummary Objective In order to maximize the efficiency and versatility of thevector-based siRNA approach, using an efficient cloning strategy we have developed anovel siRNA expression vector containing multiple tandem siRNA cassettes to observesynergistic apoptosis induction effect of it in human colon cancer cells. Methods MultipleSiRNA recombinant expression vector targeting Livin and Survivin gene simultaneouslywas constructed and transfected into human colon cancer cell. The effect of multipleSiRNA recombinant expression vector was detected by RT-PCR, Western blot, MTTreduction assay and flow cytometry. Results It was confirmed by restriction endonucleaseand sequence analysis that SiRNA recombinant expression vector targeting Livin andSurvivin gene simultaneously was constructed successfully. Livin and Survivin inhibitionratio of Livin+Survivin SiRNA at mRNA leves were 27.90% and 32.24%, Livin andSurvivin inhibition ratio at protein leves were 22.28 % and 40.86 %, growth of cancer cellswere inhibited and the apoptotic ratio was 11.69±1.37%, but the synergistic effect wasweaker than Livin and Survivin RNA interference respectively. Conclusions The multipleSiRNA recombinant expression vector targeting Livin and Survivin gene simultaneouslyhas been constructed successfully. It not only can inhibite the expression of Livin andSurvivin gene, but also can induce apoptosis in human colon cancer cells, but thesynergistic effect was weaker than Livin and Survivin RNA interference respectively.