The Biological Effects Of Antisense Oligonucleotides A Targeted Apollon On Lovo Cell Line

Objective:To screen out the antisense oligodeoxynucleotide of inhibitor of apoptosis proteins that has a better suppressing effects on the cell proliferation and promoting apoptosis on gastrointestinal tumor cells(HepG2,Lovo,MGC-803),from the IAP family members ASODN.using the selected member apollon ASODN to act on colon cancer Lovo cells,and then observe particular biochemical reactions(proliferation inhibition,inducing apoptosis,sensitivity to the chemotherapeutic drugs on the treatment of colorectal cancer, and effect on the gene expression),To provide a new target site for the treatment of colorectal cancer.Methods:Partâ… IAPs ASODN and random oligonucleotides(RODN) were transfected into Gastrointestinal cancer cells(Lovo,HepG₂,MGC-803) treated with some concentration for 48 hours,MTT assay was applied to the determination of cell proliferation,apoptotic index was examined by flow cytometry.Partâ…¡Apollon ASODN and random oligonucleotides(RODN) were transfected into Lovo cells for 48 hours,proliferation and the clone formation of Lovo cells were detected by CCK-8 and clone formation assay,respectively.The morphology was examined by fluorescence microscope.The percentage of hypodiploid cells of Lovo cells treated with apollon ASODN was determined with propidium iodide(PI) staining by flow cytometry.Early apoptosis was detected with Annexin V-FITC and PI dual parameter by flow cytometry.Furthermore,The expression of apollon gene was analyzed by real time fluorescent quantitativ reverse transcription-polymerase chain reaction.Partâ…¢Apollon ASODN and random oligonucleotides(RODN) were transfected into Lovo cells, Lovo cells were incubated treated by 5-fluorouracil(5-FU) or Epirubicin(EPI) or Cisplatin (DDP),and their combination of ASODN at different concentrations for 48 hours respectively, then the cell proliferation was detected by WST-8 test,finally their IC₅₀ and their drug-sensitivity were calculated statistically.Results:Partâ… After treated with antisense oligodeoxynucleotide targeting 48 hours,compared with blank control and random oligodeoxynucleotide groups,antisense oligodeoxynucleotide targeting IAPs can significantly suppressed the growth of gastrointestinal cancer cells and induce their apoptosis(P＜0.05),at a concentration of 0.2μmol/L,survivin ASODN has the significantly inhibited effects on the HepG₂,Lovo cells,the inhibitory rates were 84.2%,82.6%.respectively. the xiap ASODN has the significantly inhibited effects on the MGC-803 cell,inhibitory rates were 86.56%.they showed time and dose-dependent fashions.Survivin ASODN inducing the apoptosis of Lovo cell,MGC-803 cell was the strongest than others,the apoptotic rate were 34.95%,27.2%.respectively.livin ASODN inducing the apoptosis of HepG₂ cell was the strongest than others,the apoptotic rate were 22.5%.Partâ…¡After treated with antisense oligodeoxynucleotide targeting apollon 48 hours,compared with blank control and random oligodeoxynucleotide groups,antisense oligodeoxynucleotide targeting apollon can significantly suppressed the growth of colorectal cancer cells and induce their apoptosis(P＜0.05).It was found that all concentrations of the apollon ASODN used in this study showed to be able to suppress the expression of the apollon gene.antisense oligodeoxynucleotide targeting apollon induce significant increase in s-phase cells,many Lovo cells stained with Hoechst 33258 exhibited apoptotic morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation.Partâ…¢Compared with single 5-FU,EPI and DDP group,The combition of 5-FU,EPI,DDP with apollon ASODN have enhanced their chemotherapeutic effects on Lovo cells,and the sensitivity enhance to 2.5,4.47,and 5.33 times respectively.Conclusion:1.Antisense oligodeoxynucleotide targeting IAPs can inhibit the proliferation and induce the apoptosis of gastrointestinal cancer cells in vitro.2.The different antisense oligodeoxynucleotide targeting apollon can suppress the express levels of gene,and inhibit the proliferation and induce the apoptosis of colorectal cancer cells in vitro.3.0.08μmol/L apollon ASODN can enhance the chemotherapeutic drug's sensitivity of Lovo cells to 5-FU,EPI and DDP.