The Radiosensitivity Effects Of Antisense Ck13Lentivirus On Nasopharyngeal Carcinoma Cell Line Strain HNE1

Objective:This research discusses the radiosensitivity effect of Ckl3antisense gene on nasopharyngeal carcinoma cell line strain HNE1. Lentiviral vector was used to mediated transfection, transfecting anti-CK13lentivirus and empty lentivirus (pLV-EGFP) which carrying the Ckl3antisense gene to the HNE1cells and analyzing effect on cell cycle and radiationsensity of nasopharyngeal carcinoma from opposite angle. It aims to explore a safe and effective treatment of nasopharyngeal and get a breakthrough in the treatment of nasopharyngeal carcinoma progress.Methods:1.The preparation of target genes: to compound CK13antisense oligonucleotide fragments; to recombine DNA; transfection; screening.2.The conversion, amplification, restriction digestion and sequencing analysis of CK13lentivirus and empty (pLV-EGFP) which carry the Ckl3antisense gene.3.Lentivirus-mediated, transfecting CK13lentivirus and empty lentivirus (pLV-EGFP) to the HNE1cells. Naming as group Anti-CK13、group lentivirus and non-transfected cell lines HNE1named group control. It uses the methods of Realtime-PCR, Westen blotting and other molecular biology methods to measure the purpose of gene expression in cells.4.Colony Formation Assay was used to determine cell survival fraction dose, using a linear quadratic model (LQ model) and single-hit multitarget model fit cell survival curves, calculated radiobiological parameters of three groups.5.The apoptosis and Cell cycle changes were analyzed by flow cytometry.Results:l.The anti-CK13lentivirus which carrying a reasonable antisense oligonucleotide sequence5’-CACCTCCATAGCCACCTCCA-3’, which fit our study requirement. 2.CK13lentivirus and empty vector lentivirus (pLV-EGFP) which carrying the Ck13antisense gene were transfected into HNE-1. Results of RT-qPCR and Western blot showed the levels of the CK13mRNA in group A is0.284times of the levels which is the Normal cells HNE1; and the expression level reduced significantly, which difference was significant (P<0.05).3.The linear quadratic model (LQ model) and click multiple-target model shows that:after transfection of CK13antisense gene, D0, Dq, SF2were increased, a values, α/β were decreased, which indicates reducedthe gene silencing of CK13reduces the cell radiosensitivity of HNE1. After the X-ray irradiation, each group radiobiology parameters of blank cell group and negative control group were similar, indicating that the two cell radiosensitivity was no significant change. While at the same radiation dose, compared with transfected with empty vector and blank cells, the survival fraction of cells which transfected antisense CK13gene increased.4.The results of the statistics the forming clones case in each group cells after Giemsa staining indicate the formation and the formation rate of the group which is transfected CK13antisense gene is greater than the number of empty lentivirus group and blank cell group, and the difference was statistically significant (P<0.05).Conclusion:The Ck13antisense gene can obviously reduce the radiosensitivity on nasopharyngeal carcinoma cell strain line HNE1.