靶向siRNA抑制TLR9表达对放射抗拒肺癌A549细胞辐射敏感性的影响

Inhibition of TLR9 expression by siRNA influences radiosensitivity of lung cancer A549 cells

目的 旨在研究抑制放射抗拒肺癌A549的TLR9表达对其辐射敏感性的影响。方法 以放射抗拒肺癌A549（R-A549）细胞为研究对象，利用脂质体转染技术将靶向siRNA转染至R-A549细胞，Western blot验证；给予0、2、4、6和8 Gy X射线照射后，进行细胞克隆集落计数并绘制细胞存活曲线，经过单击多靶模型拟合，得到A549细胞、R-A549细胞、阴性siRNA转染细胞和TLR9 siRNA转染细胞照射的D0、Dq、N值；对4组细胞增殖能力进行测定。对R-A549细胞、阴性siRNA转染细胞和TLR9 siRNA转染细胞X射线10 Gy照射前后流式细胞周期分析，进一步说明抑制TLR9表达对R-A549细胞辐射敏感性的影响。结果 TLR9 siRNA成功抑制R-A549细胞TLR9表达，照射后其细胞克隆形成率降低，与R-A549细胞的放射增敏比（SERD0）达到1.37，而A549细胞与R-A549 细胞的SERD0为1.09；照射后TLR9 siRNA转染细胞较R-A549细胞G2/M期分布增加（t=4.323，P〈0.05），而TLR9 siRNA转染细胞G1/G0期分布少于R-A549细胞，差异有统计学意义（t=7.616，P〈0.05）。结论 siRNA可以抑制TLR9表达，降低照射后R-A549细胞的克隆形成，提高放射增敏比；照射后更多细胞发生G2/M期阻滞，同时G1/G0期细胞减少也有效抑制了照射后细胞潜在致死性损伤的修复，因此，具有辐射增敏的作用。

Objective To study the influence of TLR9 on the radiosensitivity of radiation-resistance lung cancer A549 cells （R-A549）. Methods TLR9 siRNA was transfected to R-A549 cells with liposome and TLR9 expression level was examined by Western blot. After X-ray irradiation with doses from 0 to 8 Gy, colony formation assay was performed and cell survival curve was fitted with the multi-target single-hitting model and D0, Dq, and N value of the survival of A549, R-A549, control siRNA transfected and TLR9 siRNA transfected cells was calculated. Cell proliferation ability and cell cycle distributions of these four groups were also determined. Results TLR9 siRNA successfully suppressed TLR9 expression in R-A549 cells and reduced the colony formation after irradiation with a sensitization enhancement ratio （SERD0） of 1.37 to R-A549. SERD0 of A549 to R-A549 was 1.09. After irradiation, compared to R-A549, cells in G2/M phase of TLR9 siRNA transfected population increased significantly （t=4.323, P〈0.05） but G1/G0 phase decreased significantly （t=7.616, P〈0.05）. Conclusions TLR9 siRNA increased the radiosensitivity of R-A549. Radiation induced G2/M phase arrest and G1/G0 phase decrease could suppress potential lethal damage repair and results in the enhancement of radiation sensitivity.