The Effect Of Virus-mediated Antisense CK13 Gene For Human Nasopharyngeal Carcinoma Cell HNE1 Animal Model Radiosensitivity

Purpose:Nasopharyngeal cancer (nasopharyngeal carcinoma, NPC) is a common malignancy in southern of China. At present, the main treatment for NPC is radiotherapy. The main failure of radiotherapy is due to existing local residual lesions, recurrence and distant metastasis after NPC radiotherapy. It means that a certain proportion cells (10%-20%) in NPC tissue resist the action of radiation.These cells after radiation can not only enhance the ability of resisting radiation, but also make tumors more aggressive, and contribute to the lymph nodes metastasis and distant metastasis.Therefore, it is of great importance for clinical implications to understand the mechanism of NPC radiosensitivity and explore a treatment to improve the efficacy of NPC radiosensitivity.In this study, we built a xenograft nasopharyngeal carcinoma to simulate environment in vivo with virus-mediated antisense CK13 gene cells in nude mice to provide an experimental basis for finding a more sensitive treatment of the target gene in NPC.Methods:1. The establishment of target gene contained NPC animal model A groups.(Al: HNE1 cells; A2:HNE1-lenti-NC cells; A3:HNE1-anti-CK13a cells; A4: HNE1-anti-CK13b),5 mice in each group were randomly grab.2. Injecting plasmid to NPC animal model B groups.(B1:control group,injected with saline; B2:empty plasmid group, empty lentiviral vector injection; B3:antis-ense CK13a group, Injection of antisense CK13a lentivirus; B4:antisense CK13b group, injection of antisense CK13b lentivirus),5 mice in each group were randomly grab.3.Radiotherapy, and measurement of tumor volume before and after radiotherapy.4.Killing all the mice, Weighing tumors had inhibitory rate calculation and taking specimens for pathological examination, immunohistochemistry. RT-qPCR and Western Blot.5. Analyzing the results statistically.Results:1. During the experiment,40 mice’s mortality rate is 0% and the survival rate is 100%; tumors in left armpit formed a clear visible xenograft, with an average size of about 10mm×10mm×1mm and the tumor formation rate is 100%. The two groups show xenograts are poorly differentiated squamous cell carcinoma.2. Compare the tumor size in eight groups of mice before and after irradiation.For group A, A3, A4 of tumor growth volume increased obviously compared with Al, A2,and the differences is significant(p<0.05)。while no significant difference between A1/A3 and A2/A4(p>0.05). For group B, B3, B4 of tumor growth volume increased obviously compared with B1, B2,and the differences is significant(p<0.05)。while no significant difference between B1/B3 and B2/B4(p>0.05).3. Weighing the weight of the tumor and calculate the tumor inhibition rate. For group A,A1 is the controled group and the tumor inhibition rate of A2,A3 and A4 is-0.26%、-59.64%、-63.31% respectively. The rate of the experimental group is negative.which means the tumor is heavier than the controled group and the differences is significant(p<0.05),while no significant difference betweenn A3 and A4(p>0.05).For group B,B1 is the controled group and the tumor inhibition rate of B2,B3 and B4 is 1.46%、-46.91%、-46.58% respectively. The rate of the experimental group is negative,which means the tumor is heavier than the controled group and the differences is significant(p<0.05),while no significant difference between B3 and B4(p>0.05).These results suggest that antisense CK13 can reduce the HNE1 cells radiosensitivity, thereby antagonizing the impact of radiationtherapy.3. WesternBlot and RT-qPCR showed that the expression of CK13 in the tissue of experimental groups (A3,A4,B3,B4) were significantly lower than control groups (A1,B1) and empty carrier groups (A2,B2),and the difference is significant(p<0.05).4. Wester blot and RT-qPCR results showed that while the level of the expression of CK13 changed, the level of WNT/β-catenin change, too.Conclusion:The study found that antisense CK13 combined with radiotherapy for nasopharyngeal carcinoma into HNE1 tumor volume and weight are relatively empty vector group and control group big, The inhibition rate of the experimental group and the control group show that in vivo animal models antisense CK13 reduced HNE1 cells radiosensitivity. Indirectly from the reverse proved CK13 gene may increase the radiosensitivity of nasopharyngeal carcinoma HNE1 cells. We initially found antisense CK13 desensitization effect may be related to the WNT/β-catenin signaling pathway, which provide experimental evidence to find a new target gene for NPC treatment.