Objective: To study the effects of Numb gene expression on radiation sensitivity of nonsmall cell lung cancer (NSCLC) stem cells.
Materials and methods: The side population (SP) cells A549-SP were transfected with pcDNA3.1 (pcDNA3.1 group), pcDNA-Numb (pcDNA-Numb group) and shRNA-Numb (shRNA-Numb group). Real-time quantitative polymerase chain reaction and Western blot were performed to determine Numb expression; MTT method was used to measure the proliferation activity change of the NSCLC stem cells both before and after irradiation with different doses of 60Coγ ray; Hoechst staining and Annexin V-FITC/PI were used to detect the apoptosis of the NSCLC stem cells; and colony-forming assay was used to determine the effect of Numb expression on radiation sensitivity of the NSCLC stem cells.
Results: Increased mRNA and protein expressions of the A549-SP cells were found in the pcDNA-Numb group, and decreased mRNA and protein expressions were found in the shRNA-Numb group. The optical density value of the cells decreased in the pcDNA-Numb group but increased in the shRNA-Numb group. The cells with over-expressed Numb showed obvious nuclear condensation and fragmentation; the apoptosis rate increased significantly. The cells with knockdown Numb showed less nuclear damage; the apoptosis rate significantly decreased. After irradiation, the cells in the pcDNA-Numb group showed decreased survival rate, clonality, and the values of D0, Dq, N, and SF2; whereas the cells in the shRNA-Numb group showed the opposite trend.
Conclusions: Radiation sensitivity of NSCLC stem cells was enhanced with the increase of Numb expression. Determination of Numb expression helped to evaluate the response of lung cancer to radiotherapy, which was important for guiding tumor treatment clinically.
Keywords: Numb; gene silencing; nonsmall cell lung cancer stem cells; over-expression; radiation sensitivity; stem cell apoptosis; stem cell proliferation; tumor biology.