抗MDM2小干扰RNA对辐射诱导A549肺癌细胞死亡的影响

背景与目的 在辐射反应不同的肺癌细胞A549和NCI—H446中，照射后MDM2的表达模式是明显不同的，这可能与细胞的辐射抗性有关。本研究的目的是利用靶向MDM2的小干扰RNA（smallinter—feringRNA，siRNA）验证MDM2对A549细胞辐射反应的影响。方法 利用pPUR／U6载体构建针对MDM2的质粒，采用Lipofectamine^TM 2000脂质体转染A549细胞，通过RT—PCR和Westernblot检测转染细胞MDM2表达情况，通过流式细胞仪观察转染细胞受5Gy照射后的细胞死亡情况。结果成功构建了两个抗MDM2的质粒。细胞转染72h后MDM2在A549细胞中的表达明显降低。转染siRNA后，辐射诱导的A549细胞死亡明显增加。结论MDM2可能参与了A549细胞辐射抗性的形成，靶向MDM2的siRNA可以增加辐射诱导的A549细胞死亡。

Effects of anti-MDM2 siRNA on radiation-mediated cell killing in lung cancer cell line A549

Background and objective Lung cancer cell lines A549 and NCI-H446 with different radiosensitivity to ionizing radiation (IR) have different MDM2 gene expression status, which may contribute to the radioresistance of cells. The aim of this study is to use small interfering RNA (siRNA) targeting MDM2 to investigate the influence of MDM2 gene silencing on radioresponse of A549 cell. Methods Plasmid targeting MDM2 was constructed with pPUR/U6 vector and oligonucleotide designed according to the sequence of effective antisense oligonucleotides and principles of siRNA design. A549 cells were transfected by Lipofectamine~ TM 2000. MDM2 expression in A549 cells was detected by RT-PCR and Western blot. Radiation-mediated cell killing was detected by flow cytometry. Results Two out of three siRNA plasmids were constructed successfully. siRNA transfection resulted in downregulaton of MDM2 expression of A549 cells on mRNA and protein levels. After treated with siRNA, radiation-mediated cell killing of A549 cells was significantly increased (P<0.01). Conclusion The results support the hypothesis that MDM2 gene is a candidate for radioresistance in A549 cells. siRNA targeting to MDM2 can enhance the radiation-mediated cell killing of A549 cells.