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Retinoblastoma is a childhood ocular tumor caused by inactivation of both alleles of the Rb tumor suppressor. The dynamic chromosome aberrations were observed in retinoblastoma cells. The Sleeping Beauty (SB) transposon system is a nonviral, high eficiency DNA delivery system. In this study, we aimed to investigate how transposon-mediated Rb Gene inhibits viability of Retinoblastoma cells and whether Rb inhibits cells viability by improving DNA repair pathway. The SB system includes pPiggyBac-Rb, human Rb cDNA is inserted sequentially into the pPiggyBac-vector with minimal length 5 and 3 terminal repeats of piggyBac transposon, and pPiggyBac-Helper, expresses the piggyBac transposase. Firstly, the exogenous Rb of the Sleeping Beauty Transposon-Mediated was expressed in retinoblastoma cells (SO-Rb50). Our data demonstrated SB transposon system with significantly higher transposition activity in SO-Rb50. The cells stained with Rb antibody, 100 percentage cells with pPiggyBac-Helper are Rb positive, but 68.9 percentage in cells without pPiggyBac-Helper. Analysis of integrating transfection with real time PCR indicates that the copy of Rb cDNA in SO-Rb50 with pPiggyBac-Helper increases 10.8-folds compared to the cells without pPiggyBac-Helper. Secondly, the assess end joining activity was performed to determine the role of Rb on DNA repair in SO-Rb50. The rejoining levels of Non-homologous End-joining (NHEJ) in SO-Rb50 expressing exogenous Rb were the same compared to the control. Whereas, the rejoining levels of Homologous Recombination (RH) in SO-Rb50 expressing exogenous Rb significantly increased as compared to the control. Furthermore, exogenous Rb obviously inhibits SO-Rb50 viability. As such, this study provides new insights into mechanisms of Rb in DNA repair in retinoblastoma calls. Moreover, transposon-mediated Rb Gene may become a therapeutic target for the treatment of Retinoblastoma.