Effect Of Apoptosis And Cycles Of STAT3 Antisense Oligonucleotide And Radiotherapy On Hep-2 Laryngeal Cancer Cells

Objective:To study the effect of STAT3 antisense oligonucleotide in combination with radiotherapy on proliferation, apoptosis, cells cycle, the protein expression level of STAT3 and p-STAT3 of Hep-2 cells in human laryngeal cancer, and to explore the possibility of reducing radiotherapy resistance of laryngeal carcinoma and enhancing its sensitivity to radiation dose ofγ-rays by blocking activated STAT3 signal pathway.Methods:Hep-2 cells were cultured vitro after Hep-2 cells was transfected with STAT3 SODN and STAT3 ASODN were packed with liposome Lipofectamine 2000 and transfected into laryngocarcinama cell line Hep-2 cells. Experiments were divided into STAT3 SODN group and STAT3 ASODN group and each group was either irradiated or not byγ-rays (5Gy). According to the difference of the transfection complex, the non-radiotherapy group was divided into antisense group, sense group, and blank control group. Similarly, radiotherapy group was divided into antisense+radiotherapy group, sense+radiotherapy group, and radiotherapy control group (simple radiotherapy group). MTT was used for detecting the proliferation conditions, flow cytometry was used for detecting the apoptosis condition, cell cycle condition and protein expression levels of STAT3 and p-STAT3.Results:1 Rough change of the Hep-2 cells in different groups were observed under fluorescence inversphase microscopeThe cells in blank control group are in integritged form and well growth, while the changes of cells transfected with STAT3 ASODN showed such as reducing of cell volume, anomally form, shrinking.However, the changes were more obvious than the former when the cells were treated by STAT3 ASODN in combination with radiotherapy.2 Cell proliferation conditions in Hep-2 cells treated with different concentrations STAT3 ASODN and radiotherapyThe survival rate of the Hep-2 cells in the antisense group was significant lower than that in sense and blank control groups(P<0.01), while there was no significant difference between sense and blank control group(P > 0.05). The survival rate in the antisense+radiotherapy group was significantly lower than that in the sense+radiotherapy and simple radiotherapy group(P < 0.01), while there was no significant difference between sense+radiotherapy and simple radiotherapy group(P>0.05).3 The apoptosis condition in Hep-2 cells treated with STAT3 ASODN and radiotherapyFlow cytometry showed that the apoptosis ratio of antisense+radiotherapy, sense+radiotherapy, simple radiotherapy, antisense group, sense group, and blank control group were 31.16±0.99, 10.09±0.91, 9.34±1.51, 20.10±1.12, 6.01±1.65, 4.85±1.47. The apoptosis ratio in the antisense+radiotherapy group was significantly lower than the sense+radiotherapy and simply radiotherapy group (P<0.01).There was no significant difference between sense+radiotherapy and simple radiotherapy group (P>0.05).4 Cells cycle changes in Hep-2 cells treated with STAT3 ASODN and radiotherapyFlow cytometry showed that the numbers G0/G1 phase cell decreased obviously and the rate of SPF increased in antisense+radiotherapy group, which was significantly different with the sense+radiotherapy and simply radiotherapy group(P<0.01), while there was no significant difference between sense+radiotherapy and simple radiotherapy group (P>0.05).5 Expression of STAT3 and p-STAT3 protein in Hep-2 cells treated with STAT3 ASODN and radiotherapyThe expression of STAT3 and p-STAT3 proteins in the antisense +radiotherapy were significantly lower than that in sense+radiotherapy, antisense, simple radiotherapy groups(P<0.01); while there was no significant difference between sense+radiotherapy and simple radiotherapy groups(P>0.05), but significant difference with blank control group(P<0.05).6 Correlation analysis of FI and apoptosis rate or survival fractionThrough Pearson correlation analysis, there was a negative correlation between the corresponding protein content(FI) of STAT3 and the rate of apoptosis(AR)(r=-0.996,P<0.01); while there was a positive correlation between the corresponding protein content(FI) of STAT3 and the survival fraction (SF)(r=0.937, P<0.01);there was a positive correlation between the corresponding protein content(FI) of STAT3 and the proliferation index(PI)(r=0.939, P<0.01).Conclusion:STAT3 ASODN andγ-rays can suppress the growth, induce the apoptosis and block the cell cycle. Selective inhibition of STAT3 signaling pathway may provide a new therapeutic approach for combined treatment of laryngeal cancers.