Effects Of Anti-CK13 Gene Mediated By Slow Virus In Nasopharyngeal Carcinoma Transplantation Tumor Before And After Radiotherapy

Objective:To observe and analyze the effect of anti-CK13 gene mediated by slow virus on the cell cycle and apoptosis in nasopharyngeal carcinoma HNE1 cell transplantation tumor in Nude mice before and after radiotherapy, demonstrated antisense CK13 gene has reduced susceptibility effect in the treatment of nasopharyngeal carcinoma (NPC), and provide a experimental basis for the treatment of CK13 gene in nasopharyngeal carcinoma.Methods:In this study, the first step was cell culture; the second step was animal experiment.The animal experiments were divided into O and H two groups to conduct.1. The establishment of target gene contained NPC animal model A groups.(A1: HNE1 cells; B1:HNE1-lenti-NC cells; C1:HNE1-anti-CK13a cells; D:1:HNE1- ant i-CK13b)2. Injecting plasmid to NPC animal model B groups.(A2:control group, injected with saline; B2:empty plasmid group, empty lentiviral vector injection; C2: antis-ense CK13a group, Injection of antisense CK13a lentivirus; D2:antisense CK13b group, injection of antisense CK13b lentivirus)3. Radiotherapy, and measurement of tumor volume before and after radiotherapy.4. Killing all the mice, Stripping the mice tumor tissues and take specimens for HE、Western blot、RT-qPCR and Tunel.5. Analyzing the results statistically.Results:1、During the experiment, All mice’s mortality rate is 0% and the survival rate is 100%; tumors in left armpit formed a clear visible xenograftand the tumor formation rate is 100%. The two groups show xenograts are poorly differentiated squamous cell carcinoma.2、By calculation the Volume of tumors, we found that the volume of tumor growth in the experimental group(C1、D1、C2、D2)was significantly larger compared with the control group (A1、A2) and empty vector group (B1、B2) and the differences is significant(p<0.05).3、Cell cycle detection found that A1、A2、B1 and B2 groups had no G1 phase block, but had a brief S period block, with obvious G2/M phase retardation;C1 and C2 compared with D1 and D2 we found that S phase retardation did not change significantly, but G2/M phase of block peak enhancement obviously; A1, A2 and B1, B2 groups has the same circumstance.This has a very significant statistical significance(P< 0.05).4、Tnuel detection show that cancerous tissue apoptosis rate of A1 and B1 groups are 46.52±5.55% and 45.30±9.10%, respectively.There was no significant difference compared two groups (P>0.05); But, the cancerous tissue apoptosis rate of C1 and D1 groups are 19.48±2.89% and 26.08±11.67%, There was no significant difference compared two groups (P>0.05).C1、D1 groups compare with A1 and B1 groups cancerous tissue apoptosis rate are Significantly descend (P<0.05).In H group, we found the same trend.5、Westernblotting found that the expression level of shear body of caspase-3 and PARP are significantly increased after radiation therapy in A1、A2、B1 and B2;but in the reatment group (C1、C2、D1、D2) of anti-CK13, the expression level of shear body are significantly reduce compared with The control group (A1、A2、B1、B2), This has a very significant statistical significance(P<0.05).6、RT-qPCR detect the change of the related factor mRNA change of cell cycle signal path, the results show that CK13 may closely related with ATM and Chk2 downstream signal, and control Cdc25 block in G2/M phase under the radiation conditions of nasopharyngeal carcinoma(NPC).Conclusion:Antisense oligonucleotides of CK13 gene can obviously reduce the radiation sensitivity of HNE1 nasopharyngeal carcinoma cell lines and its mechanism may through the extension of the cell cycle and reduce the apoptosis to reduce the radiation sensitivity of HNE1 cell of nasopharyngeal carcinoma (NPC), this indirect proof that CK13 gene can improve the radiation sensitivity of nasopharyngeal carcinoma (NPC) HNE1 cells;At the same time we also found that CK13 gene could regulated Cdc25c signaling pathways block nasopharyngeal carcinoma in G2/M phase under the conditions of radiation, which provide experimental evidence to be a target gene for NPC treatment.