The Biological Effects Of Antisense Oligonucleotides Targetting Livin Gene On HepG2 Cell Line

Objective:To design and chemical syntheses antisense oligodeoxynuLeotides (ASODN) of livin, investigate the biological effect of ASODN mediated by LipofectamineTM2000 on hepatocelluar carcinoma cell line HepG2, detect it's mechanisms on hepatocelluar carcinoma cells in vitro.Methods:1. Observe the mechanism of livin ASODN delivered by LipofectamineTM 2000 on HepG2:(1) The proliferating inhibition of ASODN on HepG2:quantified by WST-8 test in vitro.(2) Test of livin mRNA expression on HepG2 cell after treated with livin ASODN by RT-PCR.(3) Observation of the morphology of apoptosis by Hoechst 33258 staining.(4) Propidine iodide (PI) staining tests to observe these cells cycle changes tested by flow cytometry.(5) ANNEXIN V/PI staining test to detect the effects on apoptosis.2. The chemotherapeutic drug's sensitivity enhancement on HepG2 after treated with livin ASODN detected by WST-8 test:HepG2 cells were incubated treated by Carboplatin (CBP) or Epirubicin (EPI) and their combination of livin ASODN at different concentrations for 48 hours respectively, then the cells proliferation was detected by WST-8 test, finally their 50% inhibition concentration (IC50)and their drug-sensitivity were calcuLated statistically.ResuLts:1.(1) Both in different concentrations and different time, the growth of liver cancer cells have been inhibited in different levels, and their inhibitions have certain dose-time-effect relationships, the IC50 of livin ASODN was measured to be 204.52 nmol/L.(2) The impact on livin mRNA expression: high concentration of livin ASODN can inhibit livin mRNA expressing,400 nmol/L group inhibitory rates have reached to 52.5 percent.(3) Hoechst 33258 staining and fluorescence microscope observed:the phenomenon of cell apoptosis can scaresly be obvious in control and RODN (random oligonucleotide)group, but ASODN group are not; while the morphological changes of apoptosis can be normally seen in ASODN group: such as to be seen numerals nuclear Fragmentations.(4) PI staining: there was scare hypodiploid in control group and RODN group, while hypodiploid appeared in high dose group of livin ASODN; the rates of hypodiploid in 100 nmol/L,200 nmol/L,400 nmol/L group have reached to 13.2 percent,13.9 percent and 18.9 percent.(5) ANNEXINV/PI double staining:Apoptosis can not be obviously seen in the control group and RODN group, while the phenomenon of cells necrosis and apoptosis can be significantly seen in high concentration of livin ASODN group; the rates of apoptosis in 400 nmol/L group has reached to 18.9 percent.2. The IC50 of CBP, EPI on HepG2 cells are 22.31 mg/L and 2.40 mg/L. The IC50 of CBP or EPI combinated with livin ASODN on HepG2 cells are 6.18 mg/L and 0.19 mg/L. Compared with CBP and EPI, The combination of CBP, EPI with livin ASODN have enhanced their chemotherapeutic effect on liver cancer cells, and the sensitivity have been enhanced to 3.61 and 12.63 times.Conclusions:1. Livin antisense oligonucleotides mediated by LipofectamineTM 2000 can inhibit the proliferation of HepG2 cells.2. Livin ASODN can significantly inhibit HepG2 cells livin-mRNA expressions.3. Livin ASODN can significantly make HepG2 cells changed in the cells cycle, and also promote cells apoptosis and necrosis.4. Livin ASODN can enhance the chemotherapeutic drug's sensitivity of HepG2 cells to CBP and EPI.