The Ku80 inhibition cell model in telomerase-negative tumor cell lines U2OS and its relation to telomere and radiosensitivity / 中华放射医学与防护杂志

ABSTRACT

Objective To construct the KU80 inhibition cell model by RNAi in U2OS cell and to explore the relationship between the Ku80,telomeres and radiosensitivity in telomerase-negative tumor cells.Methods U2OS cells were transfected with the recombinant plasmids of pshRNA-K80 by the lipofectamine,and the stable transfected cell clones were selected by G418.After the selection,the cells were collected and analyzed by the flow cytometry.RT-PCR and Western blot were used to measure the expression of Ku80 and Real-time PCR was used to detect the length of telomeres.The radiosensitivity of U2OS was determined by clone formation array.Results The transfection efficiency of the positive cell clones detected by the flow cytometry was (83.23 ± 7.63) ％.The inhibition rate of the Ku80 gene transcription in the cell group with recombinant plasmid was(68.09 ± 1.16)％ and the inhibition rate of the Ku80 protein expression in the same group was (11.03 ± 2.45) ％.The results of Real-time PCR showed that the telomere length of the cell group with recombinant plasmid (1.07 ± 0.07) was significantly shorter than that of the control group (4.42 ± 1.30,F =38.58,P ＜ 0.05) and that of the empty plasmid group (4.11 ±0.84,F =38.58,P ＜ 0.05).Compared to the control group,the telomere length of the empty plasmid group did not changed(4.42 ±0.84 vs.4.11 ±0.84).U2OS cells with Ku80 expression suppressed had lower SF2 than that of the control cells (F =1089.61,P ＜0.05),and resulted in the SER of 1.47.Conclusions The Ku80 inhibition cell model in telomerase-negative U2OS cell line is successfully established which has the shorter telomere length,and is more sensitive to radiation.Telomere shortening caused by pshRNA-of Ku80 is likely to be one of the mechanisms of radiosensitization in this kind of cell model.