Juan Chai, Yongling Li, Zhongfa Jia, Chengmin Yuan, Ping Li, Tao Sun, Jianguo Niu, Huichen Wang, Jianqi Cui
Purα is a multifunctional protein that plays an important roles in DNA repair and cell cycle regulation. Purα knockout mice have been successfully established but unfortunately the knockout mice die at the 4th week after birth and that impeded the functional research of Purα in nervous system. The cells originated from nervous system is difficult for the transfection with the routine approaches and the lower transfection efficiency also hindered the research of gene manipulation in the nervous system. The lentivirus provided a new thoroughfare for the research of gene function in nervous system. On the current paper we reported here that lentivirus vector with overexpression of EGFP-Purα fusion fluorescent protein and its control-lentivirus vector with EGFP have been successfully constructed. The constructs were packaged and the human glioblastoma cell, U87MG were infected with these two lentivirus and selected with puromycin to establish the stable cell lines with overexpression of EGFP-Purα fusion protein. The expression level in the established cell lines were examined with qPCR and western blotting assay, the results demonstrated that Purα can be stably expressed in the established cell lines. The further experiments were employed to detect the effects of Purα on cell growth and proliferation with cell counting assay (CCK-8), the results proved that when the cells were treated with different concentrations of HU, the rate of cell survival in Purα overexpression group is higher than that in the control group, it suggested that Purα could protect the cells from the toxicity of HU. The protective effects of Purα on DNA damage were also evaluated with examination of the changes of the amount of DNA damage associated protein, γH2AX, as well as the pulse field gel assay, all the results illustrated that when cells were treated with 2mM HU for 18 hours, the amount of γH2AX is much less in Purα overexpression cell line than that in the control group, pulse field gel assay also exhibited the less DNA damage in Purα overexpression group than that in the control groups. Above all, these results confirmed that Purα plays a protective role in the DNA damage induced by HU.
