Radiosensitization Effect Of Shengmai Yin On Human Nasopharyngeal Carcinoma Radioresistant Strains

Background:Nasopharyngeal carcinoma（NPC）is a malignancy affected by a variety of pathogenic factors and multiple genes.Radiotherapy is the primary curative treatment for NPC.However,some patients are resistant to irradiation and will develop local recurrence or distant metastasis afterwards.The huge heterogeneity between individuals suggests that genetic differences between individuals may result in different radiosensitivity.Traditional Chinese medicine（TCM）treatment has been shown by an increasing number of studies to improve radiosensitivity and it has great application prospects for radiosensitizer.Purpose:To explore the radiosensitization effect of Shengmai Yin on human nasopharyngeal carcinoma CNE-2R cell and the mechanism of radio-sensitization by Shengmai Yin against nasopharyngeal carcinoma.Methods:In this study,CNE-2 cell radioresistance strain CNE-2R was used as the radioresistance model.The drug concentration was selected by MTT experiment,and the clonality test was used to detect radiosensitivity.The tumor-bearing mice were randomly divided into 7 groups.The maximal diameter of the transplanted tumor was measured every other day after treatment,and the tumor volume inhibition rate and the tumor inhibition rate were calculated.The DNA methylation chip was further used to detect the methylation levels of the three groups（CNE-2,CEN-2R,Shengmaiyin+CNE-2R）,and the differential genes were screened and verified.MSP and BSP experiments were used to detect the promoter methylation level of TNC gene in each group,and RT-qPCR and Western Blot experiments were used to detect the TNC gene expression level in each group.After siRNA transfection down-regulated TNC expression,the radiosensitivity and migration invasion ability of CNE-2R were detected by clone formation experiments,scratch experiments,and Trans well experiments.Result:The inhibitory effect of Shengmaiyin on CNE-2R was time-and dose-dependent.The half inhibitory concentration at 24h was 547.6ug/ml,and 48h was 135.6ug/ml.The radiosensitization ratio of CNE-2R after Shengmaiyin intervention was enhanced relative to the blank group（1.19±0.18）.The results of animal experiments showed that the volume inhibition rate（44.96%）and tumor suppression rate（37.86%）of the Shengmaiyin high-dose combined irradiation group were significantly higher than those of the simple irradiation group.DNA methylation chip test revealed that compared with sensitive strains,294 genes were hypermethylated and 595 genes were demethylated in CNE-2R cells,and 235 genes that were originally hypermethylated and 361 genes that were originally demethylated were restored to non-significantly different levels in CNE-2R cell after Shengmai Yin intervention.Compared with the sensitive strain,TNC was over-expressed in CNE-2R cells.The TNC promoter was demethylated in the CNE-2R group,and Shengmai Yin intervention could reverse its methylation level and expression level.After down-regulation of TNC,the radiosensitivity of CNE-2R cells increased,and the ability to migrate and invade was weakened.Conclusion:Shengmaiyin has radiosensitizing effect on human nasopharyngeal carcinoma CNE-2 radioresistant strains,and there is a significant difference in the genome-wide methylation status of CNE-2 cells and its radioresistant strains.Shengmaiyin can restore the methylation status of some genes in CNE-2R cell.Shengmaiyin may induce hypermethylation of TNC gene promoter region in CNE-2R cells through DNMT3a,thereby down-regulating TNC expression,affecting radiosensitivity,migration and invasion of CNE-2R cells.