Molecular Mechanism Of Stat3 Antisense Oligonucleotides In The Enhancement Of Radiosensitivity Of Hep-2 Laryngeal Carcinoma Cells

Objective: To explore the mechanism of the synergistic effection between gene therapy and radiotherapy by observing the effect of the inhibition of growth, the changes of cell cycle and the epressions of proteins of Hep-2 cells after being treated under different concentrations of ASODN and irradiation.Methods: Human laryngeal squamous cell carcinoma of Hep-2 cells were cultured in 370C, 5%CO2, 20% O2 and 95% humidity conditions in the humid temperature incubator. Cells in the logarithmic growth phase were used in the following experiment:1.To observe Hep-2 cell morphological changes with fluorescence inversphase microscope after cells were treated under transfection of stat3 ASODN, radiotherapy on 60CO and transfection addding radiotherapy.2. To detect the inhibition rate of Hep-2 cell after they were treated with different concentrations of STAT3ASODN combined with radiotherapy of 5Gy 60CO .3.Trypan blue exclusion experiment was used to count the death rate of cells under different conditions mortality.4.To measure inhibition rates of growth and the expression of stat3, p-stat3, cyclinD1, p21 and cdk4 protein in the condition of transfection of 200nmol/l stat3ASODN combined with radiotherapy of 5Gy60CO and to observe the relation between inhibition rate and the expression of protein by Flow cytometry.5. To detect the Hep-2 on the cell cycle and the expression of stat3, p-stat3, cyclinD1, p21 and cdk4 protein with different concentration of antisense ligonucleotides transfection combined quantitative radiotherapy by Flow cytometry.Results:1.The morphological changes in the condition of transfection of stat3ASODN combined with radiotherapy more significantly than cells treated under radiotherapy alone or simple ASODN transfection, the number of suspended cells increased significantly.2. Inhibition of growth in the condition of transfection of stat3ASODN combined with radiotherapy significantly greater than cells treated under radiotherapy alone or simple ASODN transfection.3.The cell death rate of Radiotherapy alone group (5Gy) and the simple ASODN transfection group (200 nmol/l) were higher than the control group, and transfection of stat3ASODN combined with radiotherapy group were significantly higher than radiotherapy alone group or simply ASODN transfection group.4.Stat3, p-stat3 protein fluorescence index of ASODN therapy group below the control group and lipid-mediated transfection group, transfection of stat3ASODN combined with radiotherapy group than radiotherapy alone group and simply ASODN transfection group down more significantly. Pearson's correlation analysis showed that stat3 and p-stat3 were significantly correlated (r=0.986 P<0.01). With the expression of stat3, p-stat3 lower the fluorescence index of cyclinD1 and cdk4 was lower, while the p21 protein fluorescence index was higher. Cdk4 protein expression and stat3, p-stat3 protein expression were positively correlated (r=0.910 r=0.949 P<0.05), P21 protein expression and stat3, p-stat3 protein expression were negatively correlated (r=-0.981 r=-0.917 P<0.05). Moreover, p21 and cdk4 showed some negatively correlation (r=-0.973 P<0.01). Pearson's correlation analysis showed that: cell inhibition rate changes were negatively correlated with stat3, p-stat3 fluorescent protein index (FI) (r =- 0.989 r=- 0.971 P<0.01).5. Flow cytometry test results showed that the cell cycle distribution after irradiation changes: with the concentration of ASODN increasing, the proportion of G0/G1 phase cells grows at the same time, cells in S phase continuously reduce. When the concentration got to 100 nmol/l, 200nmol/l, 400 nmol/l, the proportion of G0/G1 phase cells were as following: 49.69%, 66.00%, 83.94%; the proportion of cells in S phase were 42.80%, 33.05%, 10.30 %; G2/M ratio volatile. Proportion of cells in G0/G1 and ASODN concentration was positively correlated with a correlation coefficient of 0.9479 slope. When the concentration of SODN were in the 100 nmol/l and 200 nmol/l, the proportion of G0/G1 phase cells were no significantly changes, but to the concentration of 400 nmol/l, G0/G1 phase cells making up 56.3 percent; the proportion of cells in G0/G1phase and SODN concentration was no correlation (P>0.05). With ASODN concentration increasing, stat3, p-stat3 protein expression were significantly lower after irradiation, and differences between goups were statistically significant (P <0.05).Conclusion: To control the protein on the cell cycle by reducing expression of STAT3 protein after transfected Hep-2 cells with STAT3 antisense oligonucleotide, thus leading to changes in cell cycle distribution, resulting in increased sensitivity of radiotherapy of Hep-2 laryngeal carcinoma cells.