Antisense Oligodeoxyribonucleotide Target Ki-67 MRNA Inhibits Proliferation Of Human Hepatocellucar Carcinoma Cell HEPG-7402 In Vitro

ObjectiveThe aim of this study is to investigate the effect of phosphorothioate antisense oligodeoxynucleotide (ASODN) target against Ki-67 mRNA on human hepatocellular carcinoma cell HEPG-7402. And observe its function of decreasing the cell's Ki-67 gene expression and cell's proliferation in vitro. Also we are longing for finding a better method to cure hepatocellular carcinoma (HCC) by genetherapy.MethodsWe got two kinds of synthesized phosphorothioate oligodeoxynucleotides. They are antisense oligodeoxynucleotides (ASODN) and nonsense oligodeoxynucleotides (NSODN).ASODN which are 23bp long were designed to specifically hybridize with complementary target mRNA .NSODN can not hybridize with target mRNA .We defined three groups throughout the test. They are ASODN group.(cells affected by ASODN). NSODN group (cells affected by NSODN)and 1640 group(cells affected by none serum 1640). We add different drugs to the cell HEPG-7402 by their groups. By the use of MTT assay we got dates about the lowest serum density which can keep cell's normal growth. The dates were also used for drawing proliferation cure and calculating inhibit ratio. In order to observe the change of cell's appearance we use HE dyeing method .By the way of immunocytochemistry (ICC) technique we observed the changes of Ki-67 labeling index (LI). Semi-quantitative RT-PCR method was used for detect Ki-67 mRNA's change. Flow cytometric analysis (FCM) technique can observe cell phase's change. We observed the change of Ki-67 antigen by laser scanning confocal microscope (LSCM). Human tumor colonyforming assay (HTCA) assumably can simulate the condition in which cell grows in vivo. By this means we observed if ASODN can affect cell's outspread ability.Results1. Nucleolus were condensed and become more blue dyeing in the cells dealt with ASODN.2. MTT assay result shows:a) The lowest serum density which can keep cell's normal growth was 3%.b) The ASODN's IC₅₀ was about 8. 2μmol/L.c) The cell's proliferation was obviously inhibited in ASODN group(63.6%) compared with NSODN group(22.6%).d) The ASODN can inhibit cell's proliferation in the highest degree after it was use 48 hours later (P<0. 05).3. The Ki-67 labeling index in ASODN group(0. 6775 + 0. 084) is lower than other groups. (NSODN group was 0.8583 + 0. 052;1640 group was 0. 8846 ±0.085) (P<0.01).4. Semi-quantitative RT-PCR method found that the gray value of ASODN group (34.43±3.85 ) is lower than 1640 group (52.68± 7.7 ) and NSODN group (49.33 + 6.86) (P<0.01) . It shows that the ASODN inhibits the expression of 12-67 mRNA.5. By analyzing the sequence of RT-PCR product, we found it accord with the sequence reported in Genbank.6. Flow cytometric analysis (FCM) method found that the ratio of cells in Gl phase is different between groups of ASODN (88.1%),NSODN (64.1%), and 1640 (64.2%). It suggest that ASODN prevent cells from Gl phase to G2/M phase.7. Laser scanning confocal microscope (LSCM) found that the mean fluorescent intensity (MFI) is lower in ASODN group (74.71+24.37) compared with other groups (219.27+41.56 and 187.69+50.61) (P< 0.01) . The Ki-67 antigen is decreased by ASODN.8. Human tumor colonyforming assay (HTCA) found that ASODN can lower the ability of tumor cell's colonyforming and outspread growth.Conclusion1. Antisense oligodeoxyribonucleotide target Ki-67 mRNA can inhibits the proliferation of human hepatocellucar carcinoma (HCC) cell HEPG-7402 in vitro.2. The ASODN can lower the HEPG-7402 cell's Ki-67 labeling index (LI).3. The ASODN can inhibits the expression of Ki-67 mRNA.4. The ASODN can decrease the quantity of the Ki-67 antigen.5. The ASODN can lower tumor cell's colonyforming and outspread growth ability.