Research Of Kankl Gene Demethylation In Nasopharyngeal Carcinoma Cell Lines Treated With5-Aza CdR

Objective:In this study, four nasopharyngeal carcinoma cell lines were treated with5-Aza azacytidine (5-Aza-CdR) to detect the expression of Kankl gene and analyze alteration in the biological behavior of tumor cells, in order to further investigate the incidence of nasopharyngeal carcinoma mechanisms and to find new ways of treatment of nasopharyngeal carcinoma, while we take the preliminary exploration of demethylating agent5-Aza-CdR in solid tumors to provide new application in clinical ideas.Methods:Once Real-time fluorescence quantitative PCR was performed to detect the expression of Kankl genes in NPC cell lines including5-8F,6-1OB, CNE1, CNE2and normal nasopharyngeal epithelial cell line NP69. Selecting the relatively lowest expression cell lines6-10B, CNE1without any treatment in the control group and treated with different concentrations of5-Aza-CdR treatment in the experimental group. Using Real Time PCR and Western Blotting to detecte tne expression level of Kankl gene; at the same time, we used BSP cloning and sequencing assay to detecte Kankl gene promoter CpG island methylation changes; simultaneously detected the biological behavior of cellular changes including:phase contrast microscope morphological changes in each experimental group situation; using MTT assay with different concentrations of5-Aza-CdR to inspect cell proliferation; and we detect different concentrations of5-Aza-CdR treatment rate of apoptosis after72h by flow cytometry.Results:1. Kankl is downregulated in NPC cell lines5-8F,6-1OB, CNE1, CNE2compared with normal nasopharyngeal epithelial cell NP69.2The expression of Kankl had been raised with varying degrees according different concentrations of5-Aza-CdR by interventing6-1OB, CNE1. The extent of which is proportional to the concentration of the drug upregulation, Kankl expression were up-regulated or even higher than normal epithelial cells in the NP69when dealed with10μM concentrations.3. Kankl protein expression was partly recovery by Western Blotting after the treatment of10μM concentration of5-Aza-CdR within6-1OB, CNE1cells compared with untreated control group.4. BSP cloning and sequencing results showed that Kankl gene promoter region were reversed after the treatment of5-Aza-CdR with two cell lines6-1OB, CNE1, the average percentage of methylation were11.11%,13.33%, significantly lower than the drug methylation level of treatment group (respectively64.44%,64.44%, P<0.01). 5. MTT assay showed5-Aza-CdR can inhibit the growth and proliferation of6-10B, CNE1cell.6. Flow Cytometry results showed that apoptosis was significantly higher after drug’s treatment, and5-Aza-CdR can induce apoptosis.Conclusions:5-Aza-CdR can effectively reverse the aberrant methylation of Kankl gene in nasopharyngeal carcinoma cell line6-10B, CNE1, and expression of Kankl are recovery by5-Aza-CdR and it can induce tumor cell apoptosis and inhibit the growth of nasopharyngeal carcinoma cells.