| Objective: To investigate the effects of suppression of RNF8on the radiosensitivityof a human lung adenocarcinoma cell line A549, and explore possible mechanisms forthis activity.Methods: We used RNA interference technology to silence the expression of RNF8inA549cells. The expression of RNF8was detected by real-time PCR and Western blot.Clonogenic assays were used to detect whether downregulation of RNF8expressionincreased the sensitivity of A549cells to ionizing radiation. γ-H2AX foci weremeasured at different time points after radiation by immunofluorescent stanning.Expression of Ku70and Rad51were assessed by immunofluorescent stanning andWestern blot. Cell cycle and apoptosis were measured by flow cytometry (FCM)assays.Results: After transfection of RNF8siRNA, the mRNA and protein expression ofRNF8in A549cells downregulated, which led to significant decreases in cell survivalrate, D0, Dq; and the sensitivity enhancing ratio (SER) was1.54. The average numberof γ-H2AX foci per cell in RNF8siRNA transfected cells was obviously increased at0.5,2,6,24hr after radiation. Moreover, inhibition of RNF8resulted indisappearance of nuclear Rad51foci and a lower level of Rad51protein in DNAdamage response. In contrast, the number of Ku70foci and Ku70protein expressionhad no changes compared to the control group. Furthermore, we also observed aprolonged arrest at G2/M checkpoint and an unaffected apoptosis ratio upon IR whenRNF8expression was knocked down. Conclusions:1) Inhibition of RNF8enhances the radiosentivity of a lungadenocarcinoma cell line A549, which makes RNF8as an ideal radiation sensitizer forthe treatment of lung adenocarcinoma.2) Downrugulation of RNF8expressiondecreases Rad51protein level and abrogates its localization at DNA damage sites,inhibits homologous recombination repair; and also prolongs G2/M checkpoint arrest,which all suggest an enhanced radiosensitivity of A549cells induced by suppressionof RNF8. |
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