| Part Ⅰ Study on the silencing ATG5gene and the influence of the radiosensitivity of nasopharyngeal carcinoma CNE-2cells[Objective]In this part, we studyed the inhibition of expression of ATG5through RNA interference lentiviral mediated technology; observed its silencing effects on cell growth and apoptosis induced by X-ray and the sensitivity of nasopharyngeal carcinoma CNE-2cells.[Method]The ATG5gene was designed and synthesized three interference targets, and then packed slow virus, application of RT-PCR and Western-blot showed the best target effect on silence to infection to CNE-2cells, to obtain stable silencing cell line by flow sorting technology. Furthermore, CCK-8method, flow cytometry technology and colony-forming assay detected changes of radiosensitivity of nasopharyngeal carcinoma CNE-2cells after ATG5gene silencing.In the meantime, we observed ATG5gene silencing effects on the transplanted tumors growth of nude mouse induced by X-ray. Then we can study the relationship between autophagy and the radiosensitivity of nasopharyngeal carcinoma.[Result]The result of CCK-8method showed that,compared with the NC group and Control group,all groups of cells survival rate were decreased after decreasing the expression of ATG5(P<0.05). The result of flow cytometry test showed that after irradiation by6Gy ray, ATG5silencing group apoptosis index increased significantly compared with the NC group and Control group (F=394.876,P<0.01). The result of colony-forming assay showed that,compared with the Control group and NC group,silencing ATG5group survival fraction decreased significantly at each dose point (P<0.05), which means descending the expression of ATG5can increase the radiosensitivity of CNE-2cells. After irradiation by lOGy ray,compared with the Control group and NC group, the tumor growth of ATG5group was slower, and the tumor weight of ATG5group was significantly less than the other two groups (F=4.035,P<0.05).[Conclusion]Decreased expression of ATG5gene can increase the sensitivity of CNE-2cells to radiation, ATG5may play a protective role in nasopharyngeal carcinoma CNE-2cell death induced by radiation. Part II Study on the silencing PARP-1gene and the influence of the radiosensitivity of nasopharyngeal carcinoma CNE-2cells[Objective]In this part, we studyed the inhibition of expression of PARP-1through RNA interference lentiviral mediated technology; observed its silencing effects on autophagy induced by X-ray and sensitivity of nasopharyngeal carcinoma CNE-2cells. Furthermore we studyed the relationship between PARP-1and autophagy and the radiosensitivity of nasopharyngeal carcinoma, provided the theoretical basis for nasopharyngeal carcinoma radiosensitization.[Method]The PARP-1gene was designed and synthesized three interference targets, and then packed slow virus, application of RT-PCR and Western-blot showed the best target effect on silence to infection to CNE-2cells, to obtain stable silencing cell line by flow sorting technology. Western-blot experiment was used to detect CNE-2cells, which was silencing by PARP-1and ATG5gene, changes after irradiation by autophagy related protein LC3-Ⅱ, ATG5and PARP-1protein, to study the relationship between PARP-1and autophagy. Furthermore, CCK-8method,flow cytometry technology and colony-forming assay detected changes of radiosensitivity of nasopharyngeal carcinoma CNE2cells after PARP-1gene silencing.[Result]Western-blot experimental results showed that when silencing PARP-1gene irradiated by10Gy ray, the relative expression of LC3-Ⅱ decreased significantly compared with the simple irradiation group (F=34.856,P<0.01), this phenomenon also had similar performance in the silencing ATG5group. At the same time, after silencing PARP-1gene, PARP-1protein expression decreased compare with the simple irradiation group (F=14.853,P<0.01), while the expression of ATG5is also correspondingly decreased (F=10.863,P<0.01). After silencing ATG5gene which related to autophagy, ATG5protein expression decreased compare with the simple irradiation group, but the expression of PARP-1was not affected; The result of CCK-8method showed that all groups of cells survival rate were decreased after decreased the expression of PARP-1(P<0.05). The result of flow cytometry test showed that after irradiation by6Gy ray, PARP-1silencing group apoptosis index increased significantly compared with the NC group and Control group (F=501.048,P<0.01).The result of colony-forming assay showed that silencing PARP-1group survival fraction decreased significantly at each dose point (P<0.05), which means descend the expression of PARP-1can increase the radiosensitivity of CNE-2cells.[Conclusion]Radiation can induce the activation of PARP-1. PARP-1is involved in nasopharyngeal carcinoma CNE-2cells autophagy caused by ray, and may be in the upstream of corresponding signal pathway. Decreased expression of PARP-1gene can increased the sensitivity of CNE-2cells to radiation, PARP-1may play a protective role in nasopharyngeal carcinoma CNE-2cell death induced by radiation. |
PDF Full Text Request