| Objective We established two radioresistant cell lines A549R and U2OSR from A549and U2OS by repeatedly irradiating using X-ray. The radiosensitivity comparison model have the same origin and similar genetic background. The expression level of telomere repeat binding factor2(TRF2) was compared between the two modles.Methods A549cells and the U2OS cells in the exponential growth phase were repeatedly irradiated using6Mv X-ray,200cGy/d,32ds. We used colony forming assay to calculate the cell survival fraction. The expression level of TRF2mRNA and protein were measured by RT-PCR and Western blot.Results The radioresistance of A549cells and U2OS cells increased after receiving64Gy X-ray. The radioresistant cell line A549R and U2OSR were stable after30passages. For A549, the SF2and Do were0.6254±0.041and2.911±0.019. For A549R, the SF2and D0were0.7813±0.014and4.316±0.008. For U2OS, the SF2and Do were0.5459±0.009and2.851±0.012. For U2OSR, the SF2and Do were0.7081±0.013and3.846±0.007. The RT-PCR and Western blot results shows that the TRF2expression level of A549R and U2OSR were higher than A549and U2OS.Conclusion Radiosensitivity of tumor cells was significantly reduced after repeated irradiation by X-ray, which could be stable in follow-up passages. Radioresistant cell lines which have the same origin and similar genetic background were established by64Gy X-ray. They were good models for the study of the relationship between telomere binding protein TRF2and radiosensitivity. The TRF2mRNA and protein expression levels of A549R and U2OSR were both higher than A549and U2OS. Objective We constructed an interference plasmid TRF2-siRNA and transfected it to A549and U20S cells. The survival of the A549and U20S cells radiated with different dose of X-ray was measured. To analyze the innner mechanism for the radiosensitivity change of A549and U2OS, we examined telomere length, telomerase activity and cell cycle at the same time.Methods According to the the TRF2mRNA coding sequence, we designed TRF2-specific siRNA, which was transfected into A549and U2OS cells. Then the expression levels of TRF2mRNA and protein were detected by RT-PCR and Western Blot, respectively. The radiosensitivity of A549and U2OS cells was detected by clone formation assay before and after the interference by TRF2-siRNA. Telomere length and telomerase activity was detected by QPCR and telomeric repeat amplification method (TRAP-PCR-ELISA). The cell cycle of A549and U2OS were detected by flow cytometry.Results A549and U2OS cells were observed by microscope after the transfection by liposomal. RT-PCR and Western Blot showed that inhibition rate of TRF2mRNA in A549and U2OS were89%and85%and the inhibition rate of TRF2protein were75%and48%, respectively, which suggestted that the FAM-TRF2-siRNA interference sequence could reduce the expression levels of the TRF2mRNA and protein. The QPCR showed that the T/S value which stand for the telomere length of A549-NC group, A549-R group, A549-siTRF2group, A549-siTRF2-R were2.02±0.08,2.42±0.04,1.57±0.16,1.89±0.138. The telomere length of A549-R was much longer than A549-NC(P<0.05), and the telomere length of A549-siTRF2was much shorter than A549-NC(P<0.05), while the telomere length of A549-siTRF2-R were much shorter than A549-NC(P>0.05) and shorter (P<0.05) than A549-R, but longer than A549-siTRF2(P>0.05). The T/S value of U2OS-NC group, U2OS-R group, U2OS-siTRF2group, U2OS-siTRF2-R were3.64±0.18,3.00±0.33,2.77±0.12,2.04±0.10. The telomere length of U2OS-R was much shorter than U2OS-NC(P<0.05), and the telomere length of U2OS-siTRF2was much shorter than U2OS-NC(P<0.05), and the telomere length of U2OS-siTRF2-R was much shorter than U2OS-NC group, U2OS-R group, U2OS-siTRF2group(P<0.05). There was no significant difference in T/S value between the untreated group and blank group (P>0.05). TRAP-PCR-ELISA result showed that telomerase activity of A549cells was reduced from2.3554±0.1003to1.6960±0.3071after the interference(P<0.05). Cell cycle of A549and U2OS were detected by flow cytometry before and after the interference by FAM-TRF2-siRNA. The results showed that cell cycle of A549didn’t change significantly after interference but had G0/G1arrest after irradiation and S phase arrest after irradiation&interference. At the same time, there’a S and G2/M phase arrest in U2OS treated by interference or irradiation or interference&irradiation.Expression of γ H2AX in A549-siTRF2and U2OS-siTRF2were decreased.Conclusion We successfully constructed the interference sequences FAM-TRF2-siRNA, which could inhibit the expression lecel of TRF2mRNA and protein. The radiosensitivity of A549-siTRF2and U2OS-siTRF2were lower than A549and U2OS. Cells with interference on TRF2have shorter telomere which may be related with the activity of telomerase. Cell cycle of A549and U2OS were influenced by FAM-TRF2-siRNA. There were more DNA damage responses in A549-siTRF2and U2OS-siTRF2than A549and U2OS. |
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